Fusions of cytokines and tumor targeting proteins

ABSTRACT

A conjugate of a cytokine and a tumor targeting moiety (TTM) with the provisos that when cytokine is TNF-α, TNF-β or IFN-γ, the TTM is other than a CD13 ligant; when the cytokine is IL-12, the TTM is other than an antiboy to fibronectin; when the cytokine is TNF, the TTM is other than an antibody to the transferrin receptor, and when the cytokine is TNF, IFN-γ, or IL-2 the antibody is other than an antibody to the TAG72 antigen.

FIELD OF THE INVENTION

The present invention relates to a pharmaceutical composition and usesthereof.

BACKGROUND OF THE INVENTION

Tumor growth and mass represent the major limiting factor to successfulimmunotherapies. Surgical, chemio and radiation therapies areconventionally used to debulk tumors, with variable success depending onthe localization of the tumor, its diffusion and intrinsic resistance totreatments. In spite of measurable improvement in patients survival,these conventional therapies still presents conspicuous drawbacks.Debulking by surgery may be very efficient in removing the primary tumormass, but is of limited clinical utility with disseminated metastastatictumors. On the other hand, chemotherapy may be associated with the riskof selecting resistant variants, which then become untreatable.Furthermore, chemotherapy is generally very toxic for patients, and hasstrong immunosuppressive effects. For these reasons, it is necessary todevelop new approaches for cancer treatment based on differentprinciples, with low toxicity and high efficiency in eradicatingdisseminated lesions.

The antitumor activity of some cytokines is described. Some cytokineshave already been used therapeutically in humans. For example, cytokinessuch as IL-2 and IFN-γ have shown positive antitumoral activity inpatients with different types of tumors, such as kidney metastaticcarcinoma, hairy cell leukemia, Kaposi sarcoma, melanoma, multiplemieloma, and the like. Other cytokines like IFNβ, the Tumor NecrosisFactor (TNF)α, TNFβ, IL-1, 4, 6, 12, 15 and the Colony StimulatingFactors (CFSs) have shown a certain antitumoral activity on some typesof tumors.

In general, the therapeutic use of cytokines is strongly limited bytheir systemic toxicity. TNF, for example, was originally discovered forits capacity for inducing the hemorrhagic necrosis of some tumors, andfor its in vitro cytotoxic effect on different tumoral lines, but issubsequently proved to have strong pro-inflammatory activity, which can,in case of overproduction conditions, dangerously affect the human body.

As the systemic toxicity is a fundamental problem with the use ofpharmacologically active amounts of cytokines in humans, novelderivatives and therapeutic strategies are now under evaluation, aimedat reducing the toxic effects of this class of biological effectorswhile keeping their therapeutic efficacy.

Some novel approaches are directed to:

-   a) the development of fusion proteins which can deliver TNF into the    tumor and increase the local concentration. For example, the fusion    proteins consisting of TNF and tumor specific-antibodies have been    produced;-   b) the development of TNF mutants which maintain the antitumoral    activity and have a reduced systemic toxicity. Accordingly, mutants    capable of selectively recognizing only one receptor have already    been prepared;-   c) the use of anti-TNF antibodies able to reduce some toxic effects    of TNF without compromising its antitumoral activity. Such    antibodies have already been described in literature;-   d) the use of TNF derivatives with a higher half-life (for example    TNF conjugated with polyethylene glycol).

EP 251 494 discloses a system for administering a diagnostic ortherapeutic agent, which comprises: an antibody conjugated with avidinor streptavidin, an agent capable of complexing the conjugated antibodyand a compound consisting of the diagnostic or therapeutic agentconjugated with biotin, which are administered sequentially andadequately delayed, so as to allow the localization of the therapeuticor diagnostic agent through the biotin-streptavidin interaction on thetarget cell recognized by the antibody. The described therapeutic ordiagnostic agents comprise metal chelates, in particular chelates ofradionuclides and low molecular weight antitumoral agents such ascis-platinum, doxorubicin, etc.

EP 496 074 discloses a method which provides the sequentialadministration of a biotinylated antibody, avidin or streptavidin and abiotinylated diagnostic or therapeutic agent. Although cytotoxic agentslike ricin are generically mentioned, the application relative toradiolabelled compounds is mostly disclosed.

WO 95/15979 discloses a method for localizing highly toxic agents oncellular targets, based on the administration of a first conjugatecomprising the specific target molecule conjugated with a ligand or ananti-ligand followed by the administration of a second conjugateconsisting of the toxic agent bound to an anti-ligand or to the ligand.

WO 99/13329 discloses a method for targeting a molecule to tumoralangiogenic vessels, based on the conjugation of said molecule withligands of NGR receptors. A number of molecules have been suggested aspossible candidates, but doxorubicin only is specifically described. Nouse of ligands of NGR receptors as cytokines vehicles to induce immunoresponses is disclosed.

In WO01/61017 the current inventor describes how surprisingly it hasbeen found that the therapeutic index of certain cytokines can beremarkably improved and their immunotherapeutic properties can beenhanced by coupling with a ligand of the aminopeptidase-N receptor(CD13). CD13 is a transmembrane glycoprotein of 150 kDa which is highlyconserved in various species. It is expressed on normal cells as well asin myeloid tumor lines, in the angiogenic endothelium and in someepithelia. The CD13 receptor is usually identified as the “NGR”receptor, in that its peptide ligands share the amino acid “NGR” motif.

Halin C et al (2002) Nature Biotechnology 20:264-269 discloses a fusionprotein consistiing of IL-12 fused to a human antibody fragment specificto the oncofetal ED-B domain of fibronectin. Carnemolla et al (2002)Blood 99(5):1659-65 discloses a fusion protein of IL-2 and an antibodyto ED-B.

Corti A et al (1998) Cancer Research 58:3866-3872 discloses an indirectappoach or “pretargeting” approach to homing TNF to tumors comprisingtumor pre-targeting with biotintylated antibodies and avidin orstreptavidin, followed by delyaed delivery of biotinylated TNF.

Hoogenboom et al (1991) Mol. Immunol. 28:1027-1037 discloses a fusionprotein constructed by fusing part of the heavy chain gene of ananti-transferrin receptor mAb with the TNF-α gene. Yang et al (1995)Hum. Antibod. Hybrodomas 6:129-136 discloses fusing the N-terminus ofTNF with the C-terminus of the hinge region of a mAb against thetumor-associated TAG72 antigen expressed by colorectal, gastric andovarian adenocarcinoma. Yang et al (1995) Mol Immunol 32:873-881discloses the production of a monovalent Fv-TNF fusion protein with theTAG72 antigen. To our knowledge no data on the in vivo activity of theseconjugates has been reported.

Xiang et al (1993) Cancer Biother 8:327-337 discloses a recombinantbifunctional molecule of the single-chain Fv directed to TAG72 andIFN-γ; and Xiang et al (1994) Immun Cell Biol 72:275-285 disclosesdiscloses a recombinant bifunctional molecule of the single-chain Fvdirected to TAG72 and IL-2.

However, there remains a need for further and improved pharmaceuticalcompositions and methods for the treatment and diagnosis of cancer.

We have now found that the concept of targeted delivery of cytokines isbroadly applicable and surprisingly increases the therapeutic index ofchemotherapeutic drugs. Due to the complexity of the multivalentinteractions necessary for these conjugates to work (targeting receptor,TNF receptors) it is not obvious that vascular receptors different fromCD13 can work.

Statements of the Invention

According to one aspect of the present invention there is provided aconjugate of a cytokine and a tumor targeting moiety (TTM) with theprovisos that when the cytokine is TNF-α, TNF-β or IFN-γ, the TTM isother than a CD13 ligand; when the cytokine is IL-2 or IL-12, the TTM isother than an antibody to fibronectin; when the cytokine is TNF, the TTMis other than an antibody to the transferrin receptor; when the cytokineis TNF, IFN-γ or IL-2 the TTM is other than an antibody to the TAG72antigen; when the cytokine is IFN, the TTM is other than αvβ₃ integrinligand; and when the cytokine is TNF, the TTM is other than fibronectin.

In another embodiment the conjugate is not biotinylated TNF.

Preferably the cytokine is an inflammatory cytokine.

In one preferred embodiment the cytokine is a chemotherapeutic cytokine.

Preferably the cytokine is TNFα, TNFβ, IFNα, IFNβ, IFNγ, IL-1,2, 4, 6,12, 15, EMAP II, vascular endothelial growth factor (VEGF), PDGF,PD-ECGF or a chemokine.

In one embodiment the cytokine is TNF-α, TNF-β or IFN-γ.

The target compound can be expressed either on the endothelial cellssurface of tumor vessels or in the extracellular matrix in close contactwith or in the vicinity of endothelial cells.

In one embodiment the TTM is a tumor vasculature targeting moiety(TVTM).

In another embodiment the TVTM is a binding partner of a tumorvasculature receptor, marker or other extracellular component.

In another embodiment the TTM is a binding partner of a tumor receptor,marker or other extracellular component.

In another embodiment the TTM is an antibody or ligand, or a fragmentthereof. In one embodiment the TTM is contains the NGR or RGD motif, oris HIV-tat, Annexin V, Osteopontin, Fibronectin, Collagen Type I or IV,Hyaluronate, Ephrin, or is a binding partner to oncofetal fibronectin;or a fragment thereof. In one embodiment the TTM is other than HIV-tat.

In a preferred embodiment the TTM contains the NGR motif

Preferably the TTM is CNGRCVSGCAGRC, NGRAHA, GNGRG, cycloCVLNGRMEC,linear or cyclic CNGRC.

In another preferred embodiment the TTM contains the RGD motif.

In one embodiment the TTM is targeted to VEGFR, ICAM 1, 2 or 3, PECAM-1,CD31, CD13, VCAM-1, Selectin, Act R11, ActRIIB, ActRI, ActRIB, CD44,aminopeptidase A, aminopeptidase N (CD13), αvβ3 integrin, αvβ5 integrin,FGF-1, 2, 3, or 4, IL-1R, EPHR, MMP, NG2, tenascin, oncofetalfibronectin, PD-ECGFR, TNFR, PDGFR or PSMA. In another embodiment theTTM is not targeted to VEGFR.

Preferably the conjugate is in the form of a fusion protein.

In another embodiment the conjugate is in the form of nucleic acid.

According to another aspect of the present invention there is providedan expression vector comprising the nucleic acid of the presentinvention.

According to another aspect of the present invention there is provided ahost cell transformed with the expression vector of of the presentinvention.

According to another aspect of the present invention there is provided amethod for preparing a conjugate comprising culturing the host cell ofclaim under condition which provide for the expression of the conjugate.

According to yet another aspect of the present invention there isprovided a pharmaceutical composition comprising the conjugate of thepresent invention, together with a pharmaceutically acceptable carrier,diliuent or excipient.

In a preferred embodiment the composition further comprises anotherantitumor agent or diagnostic tumor-imaging compound.

Preferably the further antitumor agent is doxorubicin or melphalan.

According to a further aspect of the present invention there is provideduse of a conjugate or a pharmaceutical composition according to thepresent invention for the preparation of a medicament for treatment ordiagnosis of cancer.

Put another way, the present invention provides a method of treating ordiagnosing cancer comprising administering to a patient in need of thesame an effective amount of a conjugate or a pharmaceutical compositionaccording to the present invention.

Combinations of preferred targeting moieties and cytokines which may beused in the present invention are shown in Table A below. TABLE ACytokine Targeting Moiety IFN-α RGD-CONTAINING PEPTIDE IFN-βRGD-CONTAINING PEPTIDE IL-2 RGD-CONTAINING PEPTIDE IL-12 RGD-CONTAININGPEPTIDE EMAP II RGD-CONTAINING PEPTIDE VEGF RGD-CONTAINING PEPTIDE IL-1RGD-CONTAINING PEPTIDE IL-6 RGD-CONTAINING PEPTIDE IL-12 RGD-CONTAININGPEPTIDE PDGF RGD-CONTAINING PEPTIDE PD-ECGF RGD-CONTAINING PEPTIDE CXCchemokine RGD-CONTAINING PEPTIDE CC chemokine RGD-CONTAINING PEPTIDE Cchemokine RGD-CONTAINING PEPTIDE IL-15 RGD-CONTAINING PEPTIDE TNF-αNGR-CONTAINING PEPTIDE TNF-β NGR-CONTAINING PEPTIDE IFN-α NGR-CONTAININGPEPTIDE IFN-β NGR-CONTAINING PEPTIDE IFN-γ NGR-CONTAINING PEPTIDE IL-2NGR-CONTAINING PEPTIDE IL-12 NGR-CONTAINING PEPTIDE EMAP IINGR-CONTAINING PEPTIDE VEGF NGR-CONTAINING PEPTIDE IL-1 NGR-CONTAININGPEPTIDE IL-6 NGR-CONTAINING PEPTIDE IL-12 NGR-CONTAINING PEPTIDE PDGFNGR-CONTAINING PEPTIDE PD-ECGF NGR-CONTAINING PEPTIDE CXC chemokineNGR-CONTAINING PEPTIDE CC chemokine NGR-CONTAINING PEPTIDE C chemokineNGR-CONTAINING PEPTIDE IL-15 NGR-CONTAINING PEPTIDE TNF-α Ligand toVEGFR TNF-β Ligand to VEGFR IFN-α Ligand to VEGFR IFN-β Ligand to VEGFRIFN-γ Ligand to VEGFR IL-2 Ligand to VEGFR IL-12 Ligand to VEGFR EMAP IILigand to VEGFR VEGF Ligand to VEGFR IL-1 Ligand to VEGFR IL-6 Ligand toVEGFR IL-12 Ligand to VEGFR PDGF Ligand to VEGFR PD-ECGF Ligand to VEGFRCXC chemokine Ligand to VEGFR CC chemokine Ligand to VEGFR C chemokineLigand to VEGFR IL-15 Ligand to VEGFR TNF-α Ab to VEGFR TNF-β Ab toVEGFR IFN-α Ab to VEGFR IFN-β Ab to VEGFR IFN-γ Ab to VEGFR IL-2 Ab toVEGFR IL-12 Ab to VEGFR EMAP II Ab to VEGFR VEGF Ab to VEGFR IL-1 Ab toVEGFR IL-6 Ab to VEGFR IL-12 Ab to VEGFR PDGF Ab to VEGFR PD-ECGF Ab toVEGFR CXC chemokine Ab to VEGFR CC chemokine Ab to VEGFR C chemokine Abto VEGFR IL-15 Ab to VEGFR TNF-α HIV-tat TNF-β HIV-tat IFN-α HIV-tatIFN-β HIV-tat IFN-γ HIV-tat IL-2 HIV-tat IL-12 HIV-tat EMAP II HIV-tatVEGF HIV-tat IL-1 HIV-tat IL-6 HIV-tat IL-12 HIV-tat PDGF HIV-tatPD-ECGF HIV-tat CXC chemokine HIV-tat CC chemokine HIV-tat C chemokineHIV-tat IL-15 HIV-tat TNF-α Ligand to ICAM 1, 2 or 3 TNF-β Ligand toICAM 1, 2 or 3 IFN-α Ligand to ICAM 1, 2 or 3 IFN-β Ligand to ICAM 1, 2or 3 IFN-γ Ligand to ICAM 1, 2 or 3 IL-2 Ligand to ICAM 1, 2 or 3 IL-12Ligand to ICAM 1, 2 or 3 EMAP II Ligand to ICAM 1, 2 or 3 VEGF Ligand toICAM 1, 2 or 3 IL-1 Ligand to ICAM 1, 2 or 3 IL-6 Ligand to ICAM 1, 2 or3 IL-12 Ligand to ICAM 1, 2 or 3 PDGF Ligand to ICAM 1, 2 or 3 PD-ECGFLigand to ICAM 1, 2 or 3 CXC chemokine Ligand to ICAM 1, 2 or 3 CCchemokine Ligand to ICAM 1, 2 or 3 C chemokine Ligand to ICAM 1, 2 or 3IL-15 Ligand to ICAM 1, 2 or 3 TNF-α Ab to ICAM 1, 2 or 3 TNF-β Ab toICAM 1, 2 or 3 IFN-α Ab to ICAM 1, 2 or 3 IFN-β Ab to ICAM 1, 2 or 3IFN-γ Ab to ICAM 1, 2 or 3 IL-2 Ab to ICAM 1, 2 or 3 IL-12 Ab to ICAM 1,2 or 3 EMAP II Ab to ICAM 1, 2 or 3 VEGF Ab to ICAM 1, 2 or 3 IL-1 Ab toICAM 1, 2 or 3 IL-6 Ab to ICAM 1, 2 or 3 IL-12 Ab to ICAM 1, 2 or 3 PDGFAb to ICAM 1, 2 or 3 PD-ECGF Ab to ICAM 1, 2 or 3 CXC chemokine Ab toICAM 1, 2 or 3 CC chemokine Ab to ICAM 1, 2 or 3 C chemokine Ab to ICAM1, 2 or 3 IL-15 Ab to ICAM 1, 2 or 3 TNF-α Ligand to PECAM-1/CD31 TNF-βLigand to PECAM-1/CD31 IFN-α Ligand to PECAM-1/CD31 IFN-β Ligand toPECAM-1/CD31 IFN-γ Ligand to PECAM-1/CD31 IL-2 Ligand to PECAM-1/CD31IL-12 Ligand to PECAM-1/CD31 EMAP II Ligand to PECAM-1/CD31 VEGF Ligandto PECAM-1/CD31 IL-1 Ligand to PECAM-1/CD31 IL-6 Ligand to PECAM-1/CD31IL-12 Ligand to PECAM-1/CD31 PDGF Ligand to PECAM-1/CD31 PD-ECGF Ligandto PECAM-1/CD31 CXC chemokine Ligand to PECAM-1/CD31 CC chemokine Ligandto PECAM-1/CD31 C chemokine Ligand to PECAM-1/CD31 IL-15 Ligand toPECAM-1/CD31 TNF-α Ab to PECAM-1/CD31 TNF-β Ab to PECAM-1/CD31 IFN-α Abto PECAM-1/CD31 IFN-β Ab to PECAM-1/CD31 IFN-γ Ab to PECAM-1/CD31 IL-2Ab to PECAM-1/CD31 IL-12 Ab to PECAM-1/CD31 EMAP II Ab to PECAM-1/CD31VEGF Ab to PECAM-1/CD31 IL-1 Ab to PECAM-1/CD31 IL-6 Ab to PECAM-1/CD31IL-12 Ab to PECAM-1/CD31 PDGF Ab to PECAM-1/CD31 PD-ECGF Ab toPECAM-1/CD31 CXC chemokine Ab to PECAM-1/CD31 CC chemokine Ab toPECAM-1/CD31 C chemokine Ab to PECAM-1/CD31 IL-15 Ab to PECAM-1/CD31TNF-α Ligand to VCAM-1 TNF-β Ligand to VCAM-1 IFN-α Ligand to VCAM-1IFN-β Ligand to VCAM-1 IFN-γ Ligand to VCAM-1 IL-2 Ligand to VCAM-1IL-12 Ligand to VCAM-1 EMAP II Ligand to VCAM-1 VEGF Ligand to VCAM-1IL-1 Ligand to VCAM-1 IL-6 Ligand to VCAM-1 IL-12 Ligand to VCAM-1 PDGFLigand to VCAM-1 PD-ECGF Ligand to VCAM-1 CXC chemokine Ligand to VCAM-1CC chemokine Ligand to VCAM-1 C chemokine Ligand to VCAM-1 IL-15 Ligandto VCAM-1 TNF-α Ab to VCAM-1 TNF-β Ab to VCAM-1 IFN-α Ab to VCAM-1 IFN-βAb to VCAM-1 IFN-γ Ab to VCAM-1 IL-2 Ab to VCAM-1 IL-12 Ab to VCAM-1EMAP II Ab to VCAM-1 VEGF Ab to VCAM-1 IL-1 Ab to VCAM-1 IL-6 Ab toVCAM-1 IL-12 Ab to VCAM-1 PDGF Ab to VCAM-1 PD-ECGF Ab to VCAM-1 CXCchemokine Ab to VCAM-1 CC chemokine Ab to VCAM-1 C chemokine Ab toVCAM-1 IL-15 Ab to VCAM-1 TNF-α Ligand to Selectin TNF-β Ligand toSelectin IFN-α Ligand to Selectin IFN-β Ligand to Selectin IFN-γ Ligandto Selectin IL-2 Ligand to Selectin IL-12 Ligand to Selectin EMAP IILigand to Selectin VEGF Ligand to Selectin IL-1 Ligand to Selectin IL-6Ligand to Selectin IL-12 Ligand to Selectin PDGF Ligand to SelectinPD-ECGF Ligand to Selectin CXC chemokine Ligand to Selectin CC chemokineLigand to Selectin C chemokine Ligand to Selectin IL-15 Ligand toSelectin TNF-α Ab to Selectin TNF-β Ab to Selectin IFN-α Ab to SelectinIFN-β Ab to Selectin IFN-γ Ab to Selectin IL-2 Ab to Selectin IL-12 Abto Selectin EMAP II Ab to Selectin VEGF Ab to Selectin IL-1 Ab toSelectin IL-6 Ab to Selectin IL-12 Ab to Selectin PDGF Ab to SelectinPD-ECGF Ab to Selectin CXC chemokine Ab to Selectin CC chemokine Ab toSelectin C chemokine Ab to Selectin IL-15 Ab to Selectin TNF-α Ligand toActRII, ActRIIB, ActRI or ActRIB TNF-β Ligand to ActRII, ActRIIB, ActRIor ActRIB IFN-α Ligand to ActRII, ActRIIB, ActRI or ActRIB IFN-β Ligandto ActRII, ActRIIB, ActRI or ActRIB IFN-γ Ligand to ActRII, ActRIIB,ActRI or ActRIB IL-2 Ligand to ActRII, ActRIIB, ActRI or ActRIB IL-12Ligand to ActRII, ActRIIB, ActRI or ActRIB EMAP II Ligand to ActRII,ActRIIB, ActRI or ActRIB VEGF Ligand to ActRII, ActRIIB, ActRI or ActRIBIL-1 Ligand to ActRII, ActRIIB, ActRI or ActRIB IL-6 Ligand to ActRII,ActRIIB, ActRI or ActRIB IL-12 Ligand to ActRII, ActRIIB, ActRI orActRIB PDGF Ligand to ActRII, ActRIIB, ActRI or ActRIB PD-ECGF Ligand toActRII, ActRIIB, ActRI or ActRIB CXC chemokine Ligand to ActRII,ActRIIB, ActRI or ActRIB CC chemokine Ligand to ActRII, ActRIIB, ActRIor ActRIB C chemokine Ligand to ActRII, ActRIIB, ActRI or ActRIB IL-15Ligand to ActRII, ActRIIB, ActRI or ActRIB TNF-α Ab to ActRII, ActRIIB,ActRI or ActRIB TNF-β Ab to ActRII, ActRIIB, ActRI or ActRIB IFN-α Ab toActRII, ActRIIB, ActRI or ActRIB IFN-β Ab to ActRII, ActRIIB, ActRI orActRIB IFN-γ Ab to ActRII, ActRIIB, ActRI or ActRIB IL-2 Ab to ActRII,ActRIIB, ActRI or ActRIB IL-12 Ab to ActRII, ActRIIB, ActRI or ActRIBEMAP II Ab to ActRII, ActRIIB, ActRI or ActRIB VEGF Ab to ActRII,ActRIIB, ActRI or ActRIB IL-1 Ab to ActRII, ActRIIB, ActRI or ActRIBIL-6 Ab to ActRII, ActRIIB, ActRI or ActRIB IL-12 Ab to ActRII, ActRIIB,ActRI or ActRIB PDGF Ab to ActRII, ActRIIB, ActRI or ActRIB PD-ECGF Abto ActRII, ActRIIB, ActRI or ActRIB CXC chemokine Ab to ActRII, ActRIIB,ActRI or ActRIB CC chemokine Ab to ActRII, ActRIIB, ActRI or ActRIB Cchemokine Ab to ActRII, ActRIIB, ActRI or ActRIB IL-15 Ab to ActRII,ActRIIB, ActRI or ActRIB TNF-α Annexin V TNF-β Annexin V IFN-α Annexin VIFN-β Annexin V IFN-γ Annexin V IL-2 Annexin V IL-12 Annexin V EMAP IIAnnexin V VEGF Annexin V IL-1 Annexin V IL-6 Annexin V IL-12 Annexin VPDGF Annexin V PD-ECGF Annexin V CXC chemokine Annexin V CC chemokineAnnexin V C chemokine Annexin V IL-15 Annexin V TNF-α Ligand to CD44TNF-β Ligand to CD44 IFN-α Ligand to CD44 IFN-β Ligand to CD44 IFN-γLigand to CD44 IL-2 Ligand to CD44 IL-12 Ligand to CD44 EMAP II Ligandto CD44 VEGF Ligand to CD44 IL-1 Ligand to CD44 IL-6 Ligand to CD44IL-12 Ligand to CD44 PDGF Ligand to CD44 PD-ECGF Ligand to CD44 CXCchemokine Ligand to CD44 CC chemokine Ligand to CD44 C chemokine Ligandto CD44 IL-15 Ligand to CD44 TNF-α Ab to CD44 TNF-β Ab to CD44 IFN-α Abto CD44 IFN-β Ab to CD44 IFN-γ Ab to CD44 IL-2 Ab to CD44 IL-12 Ab toCD44 EMAP II Ab to CD44 VEGF Ab to CD44 IL-1 Ab to CD44 IL-6 Ab to CD44IL-12 Ab to CD44 PDGF Ab to CD44 PD-ECGF Ab to CD44 CXC chemokine Ab toCD44 CC chemokine Ab to CD44 C chemokine Ab to CD44 IL-15 Ab to CD44TNF-α Osteopontin TNF-β Osteopontin IFN-α Osteopontin IFN-β OsteopontinIFN-γ Osteopontin IL-2 Osteopontin IL-12 Osteopontin EMAP II OsteopontinVEGF Osteopontin IL-1 Osteopontin IL-6 Osteopontin IL-12 OsteopontinPDGF Osteopontin PD-ECGF Osteopontin CXC chemokine Osteopontin CCchemokine Osteopontin C chemokine Osteopontin IL-15 Osteopontin TNF-αFibronectin TNF-β Fibronectin IFN-α Fibronectin IFN-β Fibronectin IFN-γFibronectin IL-2 Fibronectin EMAP II Fibronectin VEGF Fibronectin IL-1Fibronectin IL-6 Fibronectin IL-12 Fibronectin PDGF Fibronectin PD-ECGFFibronectin CXC chemokine Fibronectin CC chemokine Fibronectin Cchemokine Fibronectin IL-15 Fibronectin TNF-α Collagen type I or IVTNF-β Collagen type I or IV IFN-α Collagen type I or IV IFN-β Collagentype I or IV IFN-γ Collagen type I or IV IL-2 Collagen type I or IVIL-12 Collagen type I or IV EMAP II Collagen type I or IV VEGF Collagentype I or IV IL-1 Collagen type I or IV IL-6 Collagen type I or IV IL-12Collagen type I or IV PDGF Collagen type I or IV PD-ECGF Collagen type Ior IV CXC chemokine Collagen type I or IV CC chemokine Collagen type Ior IV C chemokine Collagen type I or IV IL-15 Collagen type I or IVTNF-α Hyaluronate TNF-β Hyaluronate IFN-α Hyaluronate IFN-β HyaluronateIFN-γ Hyaluronate IL-2 Hyaluronate IL-12 Hyaluronate EMAP II HyaluronateVEGF Hyaluronate IL-1 Hyaluronate IL-6 Hyaluronate IL-12 HyaluronatePDGF Hyaluronate PD-ECGF Hyaluronate CXC chemokine Hyaluronate CCchemokine Hyaluronate C chemokine Hyaluronate IL-15 Hyaluronate TNF-αLigand to FGF-1, 2, 3 or 4 TNF-β Ligand to FGF-1, 2, 3 or 4 IFN-α Ligandto FGF-1, 2, 3 or 4 IFN-β Ligand to FGF-1, 2, 3 or 4 IFN-γ Ligand toFGF-1, 2, 3 or 4 IL-2 Ligand to FGF-1, 2, 3 or 4 IL-12 Ligand to FGF-1,2, 3 or 4 EMAP II Ligand to FGF-1, 2, 3 or 4 VEGF Ligand to FGF-1, 2, 3or 4 IL-1 Ligand to FGF-1, 2, 3 or 4 IL-6 Ligand to FGF-1, 2, 3 or 4IL-12 Ligand to FGF-1, 2, 3 or 4 PDGF Ligand to FGF-1, 2, 3 or 4 PD-ECGFLigand to FGF-1, 2, 3 or 4 CXC chemokine Ligand to FGF-1, 2, 3 or 4 CCchemokine Ligand to FGF-1, 2, 3 or 4 C chemokine Ligand to FGF-1, 2, 3or 4 IL-15 Ligand to FGF-1, 2, 3 or 4 TNF-α Ab to FGF-1, 2, 3 or 4 TNF-βAb to FGF-1, 2, 3 or 4 IFN-α Ab to FGF-1, 2, 3 or 4 IFN-β Ab to FGF-1,2, 3 or 4 IFN-γ Ab to FGF-1, 2, 3 or 4 IL-2 Ab to FGF-1, 2, 3 or 4 IL-12Ab to FGF-1, 2, 3 or 4 EMAP II Ab to FGF-1, 2, 3 or 4 VEGF Ab to FGF-1,2, 3 or 4 IL-1 Ab to FGF-1, 2, 3 or 4 IL-6 Ab to FGF-1, 2, 3 or 4 IL-12Ab to FGF-1, 2, 3 or 4 PDGF Ab to FGF-1, 2, 3 or 4 PD-ECGF Ab to FGF-1,2, 3 or 4 CXC chemokine Ab to FGF-1, 2, 3 or 4 CC chemokine Ab to FGF-1,2, 3 or 4 C chemokine Ab to FGF-1, 2, 3 or 4 IL-15 Ab to FGF-1, 2, 3 or4 TNF-α Ligand to IL-1R TNF-β Ligand to IL-1R IFN-α Ligand to IL-1RIFN-β Ligand to IL-1R IFN-γ Ligand to IL-1R IL-2 Ligand to IL-1R IL-12Ligand to IL-1R EMAP II Ligand to IL-1R VEGF Ligand to IL-1R IL-1 Ligandto IL-1R IL-6 Ligand to IL-1R IL-12 Ligand to IL-1R PDGF Ligand to IL-1RPD-ECGF Ligand to IL-1R CXC chemokine Ligand to IL-1R CC chemokineLigand to IL-1R C chemokine Ligand to IL-1R IL-15 Ligand to IL-1R TNF-αAb to IL-1R TNF-β Ab to IL-1R IFN-α Ab to IL-1R IFN-β Ab to IL-1R IFN-γAb to IL-1R IL-2 Ab to IL-1R IL-12 Ab to IL-1R EMAP II Ab to IL-1R VEGFAb to IL-1R IL-1 Ab to IL-1R IL-6 Ab to IL-1R IL-12 Ab to IL-1R PDGF Abto IL-1R PD-ECGF Ab to IL-1R CXC chemokine Ab to IL-1R CC chemokine Abto IL-1R C chemokine Ab to IL-1R IL-15 Ab to IL-1R TNF-α Ligand to CD31TNF-β Ligand to CD31 IFN-α Ligand to CD31 IFN-β Ligand to CD31 IFN-γLigand to CD31 IL-2 Ligand to CD31 IL-12 Ligand to CD31 EMAP II Ligandto CD31 VEGF Ligand to CD31 IL-1 Ligand to CD31 IL-6 Ligand to CD31IL-12 Ligand to CD31 PDGF Ligand to CD31 PD-ECGF Ligand to CD31 CXCchemokine Ligand to CD31 CC chemokine Ligand to CD31 C chemokine Ligandto CD31 IL-15 Ligand to CD31 TNF-α Ab to CD31 TNF-β Ab to CD31 IFN-α Abto CD31 IFN-β Ab to CD31 IFN-γ Ab to CD31 IL-2 Ab to CD31 IL-12 Ab toCD31 EMAP II Ab to CD31 VEGF Ab to CD31 IL-1 Ab to CD31 IL-6 Ab to CD31IL-12 Ab to CD31 PDGF Ab to CD31 PD-ECGF Ab to CD31 CXC chemokine Ab toCD31 CC chemokine Ab to CD31 C chemokine Ab to CD31 IL-15 Ab to CD31TNF-α Ligand to EPHR TNF-β Ligand to EPHR IFN-α Ligand to EPHR IFN-βLigand to EPHR IFN-γ Ligand to EPHR IL-2 Ligand to EPHR IL-12 Ligand toEPHR EMAP II Ligand to EPHR VEGF Ligand to EPHR IL-1 Ligand to EPHR IL-6Ligand to EPHR IL-12 Ligand to EPHR PDGF Ligand to EPHR PD-ECGF Ligandto EPHR CXC chemokine Ligand to EPHR CC chemokine Ligand to EPHR Cchemokine Ligand to EPHR IL-15 Ligand to EPHR TNF-α Ab to EPHR TNF-β Abto EPHR IFN-α Ab to EPHR IFN-β Ab to EPHR IFN-γ Ab to EPHR IL-2 Ab toEPHR IL-12 Ab to EPHR EMAP II Ab to EPHR VEGF Ab to EPHR IL-1 Ab to EPHRIL-6 Ab to EPHR IL-12 Ab to EPHR PDGF Ab to EPHR PD-ECGF Ab to EPHR CXCchemokine Ab to EPHR CC chemokine Ab to EPHR C chemokine Ab to EPHRIL-15 Ab to EPHR TNF-α Ephrin TNF-β Ephrin IFN-α Ephrin IFN-β EphrinIFN-γ Ephrin IL-2 Ephrin IL-12 Ephrin EMAP II Ephrin VEGF Ephrin IL-1Ephrin IL-6 Ephrin IL-12 Ephrin PDGF Ephrin PD-ECGF Ephrin CXC chemokineEphrin CC chemokine Ephrin C chemokine Ephrin IL-15 Ephrin TNF-α Ligandto MMP TNF-β Ligand to MMP IFN-α Ligand to MMP IFN-β Ligand to MMP IFN-γLigand to MMP IL-2 Ligand to MMP IL-12 Ligand to MMP EMAP II Ligand toMMP VEGF Ligand to MMP IL-1 Ligand to MMP IL-6 Ligand to MMP IL-12Ligand to MMP PDGF Ligand to MMP PD-ECGF Ligand to MMP CXC chemokineLigand to MMP CC chemokine Ligand to MMP C chemokine Ligand to MMP IL-15Ligand to MMP TNF-α Ab to MMP TNF-β Ab to MMP IFN-α Ab to MMP IFN-β Abto MMP IFN-γ Ab to MMP IL-2 Ab to MMP IL-12 Ab to MMP EMAP II Ab to MMPVEGF Ab to MMP IL-1 Ab to MMP IL-6 Ab to MMP IL-12 Ab to MMP PDGF Ab toMMP PD-ECGF Ab to MMP CXC chemokine Ab to MMP CC chemokine Ab to MMP Cchemokine Ab to MMP IL-15 Ab to MMP TNF-α Ligand to NG2 TNF-β Ligand toNG2 IFN-α Ligand to NG2 IFN-β Ligand to NG2 IFN-γ Ligand to NG2 IL-2Ligand to NG2 IL-12 Ligand to NG2 EMAP II Ligand to NG2 VEGF Ligand toNG2 IL-1 Ligand to NG2 IL-6 Ligand to NG2 IL-12 Ligand to NG2 PDGFLigand to NG2 PD-ECGF Ligand to NG2 CXC chemokine Ligand to NG2 CCchemokine Ligand to NG2 C chemokine Ligand to NG2 IL-15 Ligand to NG2TNF-α Ab to NG2 TNF-β Ab to NG2 IFN-α Ab to NG2 IFN-β Ab to NG2 IFN-γ Abto NG2 IL-2 Ab to NG2 IL-12 Ab to NG2 EMAP II Ab to NG2 VEGF Ab to NG2IL-1 Ab to NG2 IL-6 Ab to NG2 IL-12 Ab to NG2 PDGF Ab to NG2 PD-ECGF Abto NG2 CXC chemokine Ab to NG2 CC chemokine Ab to NG2 C chemokine Ab toNG2 IL-15 Ab to NG2 TNF-α Ligand to tenascin TNF-β Ligand to tenascinIFN-α Ligand to tenascin IFN-β Ligand to tenascin IFN-γ Ligand totenascin IL-2 Ligand to tenascin IL-12 Ligand to tenascin EMAP II Ligandto tenascin VEGF Ligand to tenascin IL-1 Ligand to tenascin IL-6 Ligandto tenascin IL-12 Ligand to tenascin PDGF Ligand to tenascin PD-ECGFLigand to tenascin CXC chemokine Ligand to tenascin CC chemokine Ligandto tenascin C chemokine Ligand to tenascin IL-15 Ligand to tenascinTNF-α Ab to tenascin TNF-β Ab to tenascin IFN-α Ab to tenascin IFN-β Abto tenascin IFN-γ Ab to tenascin IL-2 Ab to tenascin IL-12 Ab totenascin EMAP II Ab to tenascin VEGF Ab to tenascin IL-1 Ab to tenascinIL-6 Ab to tenascin IL-12 Ab to tenascin PDGF Ab to tenascin PD-ECGF Abto tenascin CXC chemokine Ab to tenascin CC chemokine Ab to tenascin Cchemokine Ab to tenascin IL-15 Ab to tenascin TNF-α Ligand to PD-ECGFRTNF-β Ligand to PD-ECGFR IFN-α Ligand to PD-ECGFR IFN-β Ligand toPD-ECGFR IFN-γ Ligand to PD-ECGFR IL-2 Ligand to PD-ECGFR IL-12 Ligandto PD-ECGFR EMAP II Ligand to PD-ECGFR VEGF Ligand to PD-ECGFR IL-1Ligand to PD-ECGFR IL-6 Ligand to PD-ECGFR IL-12 Ligand to PD-ECGFR PDGFLigand to PD-ECGFR PD-ECGF Ligand to PD-ECGFR CXC chemokine Ligand toPD-ECGFR CC chemokine Ligand to PD-ECGFR C chemokine Ligand to PD-ECGFRIL-15 Ligand to PD-ECGFR TNF-α Ab to PD-ECGFR TNF-β Ab to PD-ECGFR IFN-αAb to PD-ECGFR IFN-β Ab to PD-ECGFR IFN-γ Ab to PD-ECGFR IL-2 Ab toPD-ECGFR IL-12 Ab to PD-ECGFR EMAP II Ab to PD-ECGFR VEGF Ab to PD-ECGFRIL-1 Ab to PD-ECGFR IL-6 Ab to PD-ECGFR IL-12 Ab to PD-ECGFR PDGF Ab toPD-ECGFR PD-ECGF Ab to PD-ECGFR CXC chemokine Ab to PD-ECGFR CCchemokine Ab to PD-ECGFR C chemokine Ab to PD-ECGFR IL-15 Ab to PD-ECGFRTNF-α Ligand to TNFR TNF-β Ligand to TNFR IFN-α Ligand to TNFR IFN-βLigand to TNFR IFN-γ Ligand to TNFR IL-2 Ligand to TNFR IL-12 Ligand toTNFR EMAP II Ligand to TNFR VEGF Ligand to TNFR IL-1 Ligand to TNFR IL-6Ligand to TNFR IL-12 Ligand to TNFR PDGF Ligand to TNFR PD-ECGF Ligandto TNFR CXC chemokine Ligand to TNFR CC chemokine Ligand to TNFR Cchemokine Ligand to TNFR IL-15 Ligand to TNFR TNF-α Ab to TNFR TNF-β Abto TNFR IFN-α Ab to TNFR IFN-β Ab to TNFR IFN-γ Ab to TNFR IL-2 Ab toTNFR IL-12 Ab to TNFR EMAP II Ab to TNFR VEGF Ab to TNFR IL-1 Ab to TNFRIL-6 Ab to TNFR IL-12 Ab to TNFR PDGF Ab to TNFR PD-ECGF Ab to TNFR CXCchemokine Ab to TNFR CC chemokine Ab to TNFR C chemokine Ab to TNFRIL-15 Ab to TNFR TNF-α Ligand to PDGFR TNF-β Ligand to PDGFR IFN-αLigand to PDGFR IFN-β Ligand to PDGFR IFN-γ Ligand to PDGFR IL-2 Ligandto PDGFR IL-12 Ligand to PDGFR EMAP II Ligand to PDGFR VEGF Ligand toPDGFR IL-1 Ligand to PDGFR IL-6 Ligand to PDGFR IL-12 Ligand to PDGFRPDGF Ligand to PDGFR PD-ECGF Ligand to PDGFR CXC chemokine Ligand toPDGFR CC chemokine Ligand to PDGFR C chemokine Ligand to PDGFR IL-15Ligand to PDGFR TNF-α Ab to PDGFR TNF-β Ab to PDGFR IFN-α Ab to PDGFRIFN-β Ab to PDGFR IFN-γ Ab to PDGFR IL-2 Ab to PDGFR IL-12 Ab to PDGFREMAP II Ab to PDGFR VEGF Ab to PDGFR IL-1 Ab to PDGFR IL-6 Ab to PDGFRIL-12 Ab to PDGFR PDGF Ab to PDGFR PD-ECGF Ab to PDGFR CXC chemokine Abto PDGFR CC chemokine Ab to PDGFR C chemokine Ab to PDGFR IL-15 Ab toPDGFR TNF-α Ligand to PSMA TNF-β Ligand to PSMA IFN-α Ligand to PSMAIFN-β Ligand to PSMA IFN-γ Ligand to PSMA IL-2 Ligand to PSMA IL-12Ligand to PSMA EMAP II Ligand to PSMA VEGF Ligand to PSMA IL-1 Ligand toPSMA IL-6 Ligand to PSMA IL-12 Ligand to PSMA PDGF Ligand to PSMAPD-ECGF Ligand to PSMA CXC chemokine Ligand to PSMA CC chemokine Ligandto PSMA C chemokine Ligand to PSMA IL-15 Ligand to PSMA TNF-α Ab to PSMATNF-β Ab to PSMA IFN-α Ab to PSMA IFN-β Ab to PSMA IFN-γ Ab to PSMA IL-2Ab to PSMA IL-12 Ab to PSMA EMAP II Ab to PSMA VEGF Ab to PSMA IL-1 Abto PSMA IL-6 Ab to PSMA IL-12 Ab to PSMA PDGF Ab to PSMA PD-ECGF Ab toPSMA CXC chemokine Ab to PSMA CC chemokine Ab to PSMA C chemokine Ab toPSMA IL-15 Ab to PSMA TNF-α Vitronectin TNF-β Vitronectin IFN-αVitronectin IFN-β Vitronectin IFN-γ Vitronectin IL-2 Vitronectin IL-12Vitronectin EMAP II Vitronectin VEGF Vitronectin IL-1 Vitronectin IL-6Vitronectin IL-12 Vitronectin PDGF Vitronectin PD-ECGF Vitronectin CXCchemokine Vitronectin CC chemokine Vitronectin C chemokine VitronectinIL-15 Vitronectin TNF-α Laminin TNF-β Laminin IFN-α Laminin IFN-βLaminin IFN-γ Laminin IL-2 Laminin IL-12 Laminin EMAP II Laminin VEGFLaminin IL-1 Laminin IL-6 Laminin IL-12 Laminin PDGF Laminin PD-ECGFLaminin CXC chemokine Laminin CC chemokine Laminin C chemokine LamininIL-15 Laminin TNF-α Ligand to oncofetal fibronectin TNF-β Ligand tooncofetal fibronectin IFN-α Ligand to oncofetal fibronectin IFN-β Ligandto oncofetal fibronectin IFN-γ Ligand to oncofetal fibronectin IL-2Ligand to oncofetal fibronectin IL-12 Ligand to oncofetal fibronectinEMAP II Ligand to oncofetal fibronectin VEGF Ligand to oncofetalfibronectin IL-1 Ligand to oncofetal fibronectin IL-6 Ligand tooncofetal fibronectin IL-12 Ligand to oncofetal fibronectin PDGF Ligandto oncofetal fibronectin PD-ECGF Ligand to oncofetal fibronectin CXCchemokine Ligand to oncofetal fibronectin CC chemokine Ligand tooncofetal fibronectin C chemokine Ligand to oncofetal fibronectin IL-15Ligand to oncofetal fibronectin TNF-α Ab to oncofetal fibronectin TNF-βAb to oncofetal fibronectin IFN-α Ab to oncofetal fibronectin IFN-β Abto oncofetal fibronectin IFN-γ Ab to oncofetal fibronectin EMAP II Ab tooncofetal fibronectin VEGF Ab to oncofetal fibronectin IL-1 Ab tooncofetal fibronectin IL-6 Ab to oncofetal fibronectin PDGF Ab tooncofetal fibronectin PD-ECGF Ab to oncofetal fibronectin CXC chemokineAb to oncofetal fibronectin CC chemokine Ab to oncofetal fibronectin Cchemokine Ab to oncofetal fibronectin IL-15 Ab to oncofetal fibronectin

It will be appreciated that in the above Table the term “Ab” representsantibody, and that the antibodies and ligands include fragments thereof.

In particularly preferred embodiments the conjugate comprises TNF-α orTNF-β and an NGR-containing peptide, or TNF-α or TNF-β and anRGD-containing peptide.

In a preferred embodiment the conjugate is in the form of a fusionprotein.

In another aspect of the present invention there is provided apharmaceutical composition comprising an effective amount of aconjugation product of TNF and a first TTM or a polynucleotide encodingthe same, and an effevctive amount of IFN-γ and a second TTM or apolynucleotide encoding the same, wherein said first TTM and saidsecoond TTM compete for different receptors.

Some Key Advantages of the Invention

To reach cancer cells in solid tumors, chemotherapeutic drugs must enterthe tumor blood vessels, cross the vessel wall and finally migratethrough the interstitium. Heterogeneous tumor perfusion, vascularpermeability and cell density, and increased interstitial pressure couldrepresent critical barriers that may limit the penetration of drugs intoneoplastic cells distant to from tumor vessels and, consequently, theeffectiveness of chemotherapy (1). Strategies aimed at improving drugpenetration in tumors are, therefore, of great experimental and clinicalinterest.

A growing body of evidence suggests that Tumor Necrosis Factor-α (TNF),and inflammatory cytokine endowed with potent anti-tumor activity, couldbe exploited for this purpose. For example, the addition of TNF toregional isolated limb perfusion with melphalan or doxorubicin hasproduced higher response rates in patients with extremity soft-tissuesarcomas or melanomas than those obtained with chemotherapeutic drugsalone (2-6). TNF-induced alteration of the endothelial barrier function,reduction of tumor interstitial pressure, increased chemotherapeuticdrug penetration and tumor vessel damage are believed to be importantmechanisms of the synergy between TNF and chemotherapy (3, 4, 7-10).Unfortunately, systemic administration of TNF is accompanied byprohibitive toxicity, the maximum tolerated dose (8-10 μg/kg) being10-50 times lower than the estimated effective dose (11, 12). For thisreason, systemic administration of TNF has been abandoned and theclinical use of this cytokine is limited to locoregional treatments.Nevertheless, some features of the TNF activity, in particular theselectivity for tumor-associated vessels and the synergy withchemotherapeutic drugs, has continued to nourish hopes as regards thepossibility of wider therapeutic applications (13).

The vascular effects of TNF provide the rational for developing a“vascular targeting” strategy aimed at increasing the local efficacy andat enabling systemic administration of therapeutic doses. We have shownrecently that targeted delivery of TNF to tumor vessels can be achievedby coupling this protein with the CNGRC peptide, an aminopeptidase N(CD13) ligand that targets the tumor neovasculature (14). In the presentwork, we have investigated whether vascular targeting with otherconjugates could enhance the penetration of chemotherapeutic drugs intumors and improve their efficacy. In addition, we look at whethervascular targeting with the conjugates can reduce drug-penetrationbarriers and increase the amount of chemotherapeutics that reach cancercells.

To reduce tumor cells to a number that can be completely destroyed byanti-tumor effector T cells, we must envisage a way to debulk tumormasses in a way that, unlike chemotherapy, is not immunosuppressive.

In this respect, we believe targeting tumor vessels to kill tumor cellsappears to be one of the most promising therapeutic approach for cancer.Tumor-induced vascular endothelium is composed of non-transformed cells,which are therefore not subjected to mutations induced by therapy. Thus,repeated treatments that target tumor vascular endothelium could inprinciple be administered, without running into the danger of selectingfor resistant variants. Second, by destroying a relatively low number oftumor vessels, it may be possible to destroy a huge number of tumorcells, which rely on blood support to thrive.

A biological therapy that impairs the function of tumor-associatedvessel and disrupt new vessel formation without causingimmunosuppression would be, therefore, a very attractive approach todebulk tumor masses prior immunotherapy or other therapeuticinterventiions.

Among the various cytokines and biological response modifiers that canaffect tumor vessels as well as the immune system, TNF-α, alone or incombination with interferon gamma and chemotherapy is undoubtedly one ofthe more potent. The massive haemorragic necrosis and tumor shrinkagethat this cytokine can induce within 24 hours in animal tumors is wellrecognized since its discovery. It is now well established that TNF candisrupt the tumor macro- and microvasculature of metastatic melanomas ofthe extremities also in patients, when regionally administered at highdoses in combination with interferon gamma and melphalan, by isolatedlimb perfusion. TNF can cause a cascade of events leading to endothelialcell damage, platelet aggregation, intravascular fibrin deposition andcoagulation, and culminating in the arrest of the tumor circulation.Remarkaly, normal vessels close to the tumor remain unaffectedindicating that TNF can somehow distinguish the vasculature of normaltissues from that of tumors. One attractive possibility is therefore toexploit TNF to induce tumor debulking prior other therapeuticintervention.

Another potential advantage of tumor debulking with TNF overconventional chemotherapeutic agents is that it is not animmunosuppressor, but on the contrary, it is an important activator ofthe immune response. Indeed TNF can activate antigen presenting cellwhich in turn are important key mediator of the immune response, as wellas a variety of other mechanisms that contribute to an efficientimmuneresponse.

Unfortunately, the clinical use of TNF as an anticancer drug has beenlimited so far to loco-regional treatments because of dose-limitingsystemic toxicity and poor therapeutic index.

Soluble, bioactive TNF is a homotrimeric protein that slowly dissociatesinto inactive, monomeric subunits at picomolar levels (1). Biologicalactivities are induced by trimeric TNF upon interaction with andsubsequent homotypic clustering of two distinct cell surface receptors(2) of 55-60 kDa and 75-80 kDa, respectively (p55TNFR and p75TNFR). Thep55TNFR is thought to mediate most TNF effects (3, (4, (5, (6, (7),whereas the p75TNFR, due to its higher affinity (K_(d)=0.1×10⁻⁹ M vs.0.5×10⁻⁹ M for p55TNFR), plays an important role in increasing the localconcentration of TNF and in passing the ligand to the p55TNFR (8, (9).Besides these supportive or modulating effects, direct signalling by thep75TNFR can also contribute to several cellular responses, likeproliferation of thymocytes, fibroblasts and natural killer cells,GM-CSF secretion (2, (10, (11), and in determining locally restrictedresponses induced by the endogenous membrane-bound form of TNF (12).

Clinical trials performed to demonstrate anti-tumour efficacy of TNFshowed that administration of large, therapeutically effective doses ofTNF were accompanied by unacceptably high levels of systemic toxicity,the dose-limiting toxicity being usually hypotension. Therefore,attempts to administer TNF, systemically, to tumour patients, have beenessentially discontinued. Nevertheless, the remarkable anti-tumouractivity of TNF in some animal models has continued to nourish hopes asregards the possibility of a therapeutic application of TNF in humans.This implied, however, the need to find ways to reduce TNF toxicity uponsystemic administration or to deliver TNF with relative or absoluteselectivity to the actual therapeutic target—the tumour.

The maximum tolerated dose of bolus TNF (intravenous) in humans is218-410 μg/m² (28), about 10-fold lower than the effective dose inanimals (29). Based on data from murine models it is believed that atleast 10 times higher dose is necessary to achieve anti-tumor effects inhumans.

One approach that has been pursued in order to exploit antitumouractivities of TNF, while avoiding its systemic toxicity, has beenregional or local administration. Thus, local administration of TNF hasshown promising response rates in Kaposi's sarcoma, plasmacytomas,ovarian adenocarcinomas and various metastatic tumours in the liver (30,(31). As regards regional administration, striking results have beenobtained when high doses of TNF were used in combination with melphalanin isolated limb perfusion to treat extremity melanoma and sarcoma. Thisprotocol has allowed to achieve 90-100% complete response rates withtumours undergoing haemorragic necrosis 32), an observation consistentwith those from preclinical studies in some experimental animal tumourmodels.

Although these results are encouraging, the applicability of theseapproaches is likely to remain limited for two main reasons. First, inmost instances where locoregional therapy can be envisaged it is likelythat, also in the future, the use of other established means ofintervention (e.g. surgery, radiotherapy) will prevail. Second, bydefinition, malignancies tend do disseminate and it is in this setting,where locoregional therapy is precluded, that the medical need for newtherapeutic approaches is most acute. In the first clinical study onhyperthermic isolated-limb perfusion, high response rates were obtainedwith the unique dose of 4 mg of TNF in combination with melphalan andinterferon-γ (32). Other works showed that interferon-γ can be omittedand that even lower doses of TNF can be sufficient to induce atherapeutic response (33, (34). Since also these treatments are notdevoid of risk of toxicity (35), the vascular targeting with TNFderivatives may represent an alternative approach to reduce toxiceffects also in this setting.

DETAILED DESCRIPTION

Various preferred features and embodiments of the present invention willnow be described by way of non-limiting example.

Although in general the techniques mentioned herein are well known inthe art, reference may be made in particular to Sambrook et al.,Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al., ShortProtocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons, Inc (aswell as the complete version Current Protocols in Molecular Biology).

Conjugate

The present invention relates to a conjugate which is a moleculecomprising at least one targeting moiety/polypeptide linked to at leastcytokine formed through genetic fusion or chemical coupling. By “linked”we mean that the first and second sequences are associated such that thesecond sequence is able to be transported by the first sequence to atarget cell. Thus, conjugates include fusion proteins in which thetransport protein is linked to a cytokine via their polypeptidebackbones through genetic expression of a DNA molecule encoding theseproteins, directly synthesised proteins and coupled proteins in whichpre-formed sequences are associated by a cross-linking agent. The termis also used herein to include associations, such as aggregates, of thecytokine with the targeting protein. According to one embodiment thesecond sequence may comprise a polynucleotide sequence. This embodimentmay be seen as a protein/nucleic acid complex.

The second sequence may be from the same species as the first sequence,but is present in the conjugate of the invention in a manner differentfrom the natural situation, or from a different species.

The conjugates of the present invention are capable of being directed toa cell so that an effector function corresponding to the polypeptidesequence coupled to the transport sequence can take place.

The peptide can be coupled directly to the cytokine or indirectlythrough a spacer, which can be a single amino acid, an amino acidsequence or an organic residue, such as6-aminocapryl-N-hydroxysuccinimide.

The peptide ligand is preferably linked to the cytokine N-terminus thusminimising any interference in the binding of the modified cytokine toits receptor. Alternatively, the peptide can be linked to amino acidresidues which are amido- or carboxylic-bond acceptors, which may benaturally occurring on the molecule or artificially inserted usinggenetic engineering techniques. The modified cytokine is preferablyprepared by use of a cDNA comprising a 5′-contiguous sequence encodingthe peptide.

According to a preferred embodiment, there is provided a conjugationproduct between TNF and the CNGRC sequence in which the amino-terminalof TNF is linked to the CNGRC peptide through the spacer G (glycine).

Cytokines

Drug penetration into neoplastic cells is critical for the effectivenessof solid-tumor chemotherapy. To reach cancer cells in solid tumors,chemotherapeutic drugs must enter the drug blood vessels, cross thevessel wall and finally migrate through the interstitium. Heterogeneoustumor perfusion, vascular permeability and cell density, and increasedinterstitial pressure may represent critical barriers that may limit thepenetration of drugs into neoplastic cells and, consequently, theeffectiveness of chemotherapy. Cytokines which have the effect ofaffecting these factors are therefore useful in the present invention. Anon-limiting list of cytokines which may be used in the presentinvention is: TNFα, TNFβ, IFNα, IFNβ, IFNγ, IL-1,2, 4, 6, 12, 15, EMAPII, vascular endothelial growth factor (VEGF), PDGF, PD-ECGF or achemokine.

TNF

TNF acts as an inflammatory cytokine and has the effect of inducingalteration of the endothelial barrier function, reducing of tumorinterstitial pressure, and increasing chemotherapeutic drug penetrationand tumor vessel damage.

The first suggestion that a tumor necrotizing molecule existed was madewhen it was observed that cancer patients occasionally showedspontaneous regression of their tumors following bacterial infections.Subsequent studies in the 1960s indicated that host-associated (orendogenous) mediators, manufactured in response to bacterial products,were likely responsible for the observed effects. In 1975 it was shownthat a bacterially-induced circulating factor had strong anti-tumoractivity against tumors implanted in the skin in mice. This factor,designated tumor necrosis factor (TNF), was subsequently isolated,cloned, and found to be the prototype of a family of molecules that areinvolved with immune regulation and inflammation. The receptors for TNFand the other members of the TNF superfamily also constitute asuperfamily of related proteins.

TNF-related ligands usually share a number of common features. Thesefeatures do not include a high degree of overall amino acid (aa)sequence homology. With the exception of nerve growth factor (NGF) andTNF-beta, all ligands are synthesised as type II transmembrane proteins(extracellular C-terminus) that contain a short cytoplasmic segment(10-80 aa residues) and a relatively long extracellular region (140-215aa residues). NGF, which is structurally unrelated to TNF, is includedin this superfamily only because of its ability to bind to the TNFRSFlow affinity NGF receptor (LNGFR). NGF has a classic signal sequencepeptide and is secreted. TNF-β, in contrast, although also fullysecreted, has a primary structure much more related to type IItransmembrane proteins. TNF-β might be considered as a type II proteinwith a non-functional, or inefficient, transmembrane segment. Ingeneral, TNFSF members form trimeric structures, and their monomers arecomposed of beta-strands that orient themselves into a two sheetstructure. As a consequence of the trimeric structure of thesemolecules, it is suggested that the ligands and receptors of the TNSFand TNFRSF superfamilies undergo “clustering” during signaltransduction.

TNF-α Human TNF-α is a 233 aa residue, nonglycosylated polypeptide thatexists as either a transmembrane or soluble protein. When expressed as a26 kDa membrane bound protein, TNF-α consists of a 29 aa residuecytoplasmic domain, a 28 aa residue transmembrane segment, and a 176 aaresidue extracellular region. The soluble protein is created by aproteolytic cleavage event via an 85 kDa TNF-alpha converting enzyme(TACE), which generates a 17 kDa, 157 aa residue molecule that normallycirculates as a homotrimer.

TNF-β/LT-α: TNF-β, otherwise known as lymphotoxin-α (LT-α) is a moleculewhose cloning was contemporary with that of TNF-α. Although TNF-βcirculates as a 171 aa residue, 25 kDa glycosylated polypeptide, alarger form has been found that is 194 aa residues long. The human TNF-βcDNA codes for an open reading frame of 205 aa residues (202 in themouse), and presumably some type of proteolytic processing occurs duringsecretion. As with TNF-α, circulating TNF-β exists as a non-covalentlylinked trimer and is known to bind to the same receptors as TNF-α.

In one embodiment the TNF is a mutant form of TNF capable of selectivelybinding to one of the TNF receptors (Loetscher H et al (1993) J BiolChem 268:26350-7; Van Ostade X et al (1993) Nature 361:266-9).

Several approaches aimed at reducing systemic toxicity of TNF whilepreserving its antitumour activity have been pursued. Although the finalgoal is the same as that in the previous section, i.e. an increase ofthe therapeutic index, the rationale is significantly different. In theprevious case, a generalised enhancement of a single biologicalactivity, cytotoxicity, initially thought to represent an in vitrocorrelate of the anti-tumour activity of TNF, in the present a selectivemodification of the biological profile of TNF leading to thepreservation of some activities and to the loss of others.

Work along this latter rationale took advantage, mostly, of thepossibility to engineer TNF mutants binding to only one of the two TNFR.Efforts in this direction were initiated by the observation that humanTNF binds only one (p55TNFR) of the two mouse TNFR, the interaction withthe mouse p75TNFR being species-specific (2). In vivo studies showedthat systemic toxicity of human TNF was approximately 50 times lowerthan that of mouse TNF when administered to normal mice, whileanti-tumour activity was equivalent (44). These observations suggestedthat TNF mutants that maintained binding to the p75TNFR might have amore favourable therapeutic index than natural TNF. Indeed, suchreceptor-selective TNF mutants were subsequently obtained throughsite-directed mutagenesis approaches (4, (45). Studies performed with ap55TNFR-specific mutant showed that it was as effective as natural TNFwith regard to in vivo antitumour activity, whereas activities onneutrophils and endothelial cells, two cell types believed to play animportant part in TNF-induced systemic toxicity, were greatly decreased(6).

Although these results were highly encouraging in view of a possibletherapeutic use of these mutants in anti-tumour therapy, hopes that hadbeen raised were considerably dampened by the observation that inprimates also the p55TNFR plays an important role in systemic toxicity(46) and that the gain in terms of reduced toxicity was lost when themutants were administered in combination with an agent that increasedsensitivity to TNF, like IL-1, LPS or, most importantly in this setting,in the presence of the tumour itself, which sensitises the organism toTNF in a manner similar to that described for the exogenouslyadministered substances previously referred to (47).

In view of the above we teach that coupling these or other TNF muteinswith an alpha v beta 3 ligand may result in an improvement of theirtherapeutic index.

Many other inflammatory cytokines also have the property of increasingendothelial vessel permeability, and it will be appreciated that theinvention can be applied to such cytokines, together with agents whichincrease expression of such cytokines. Inflammatory cytokines, alsoknown as pro-inflammatory cytokines, are a number of polypeptides andglycoproteins with molecular weights between 5 kDa and 70 kDa. They havea stimulating effect on the inflammatory response. The most importantinflammatory cytokines are TNF, IL-1, IL-6 and IL-8.

A Table of some cytokines classified as inflammatory cytokines is shownbelow: Inflammatory Cytokines Group Individual cytokines Endogenouscytokines IL-1, TNF-α, IL-6 Up-regulation IL-1, TNF-α, IL-6, IFN-α,INF-β, chemokines Stimulation of the production IL-1, IL-6, IL-11,TNF-α, INF-γ, TGF-β, of acute phase reactants LIF, OSM, CNTFChemoattractant cytokines CXC chemokines IL-8, PF-4, PBP, NAP-2, β-TG CCchemokines MIP-1α, MIP-1β, MCP-1, MCP-2, MCP- 3, RANTES C chemokinesLymphotactin Stimulation of inflammatory IL-12 cytokines

TGF-β: transforming growth fadtor, LIF: leukemia inhibitory factor; OSM:oncostatin M; CNTF: ciliary neurotrophic factor; PF-4: platelet factor4; PBP: platelet basic protein; NAP-2: neutrophil activating protein 2;β-TG: β-thromboglobulin; MIP: macrophage inflammatory protein; MCP:monocyte chemoattractant protein.

The up-regulation of inflammatory response is also performed by IL-11,IFN-α, IFN-β, and especially by the members of the chemokinesuperfamily. TGF-β in some situations has a number of inflammatoryactivities including chemoattractant effects on neutrophils, Tlymphocytes and inactivated monocytes.

IL-2

Because of the central role of the IL-2/IL-2R system in mediation of theimmune and inflammatory responses, it is obvious that monitoring andmanipulation of this system has important diagnostic and therapeuticimplications. IL-2 has shown promise as an anti-cancer drug by virtue ofits ability to stimulate the proliferation and activities oftumor-attacking LAK and TIL (tumor-infiltrating lymphocytes) cells.However, problems with IL-2 toxicity are still of concern and meritinvestigation. The present invention addresses this problem.

IL-15

Interleukin 15 (IL-15) is a novel cytokine that shares many biologicalproperties with, but lacks amino acid sequence homology to, IL-2. IL-15was originally identified in media conditioned by a monkey kidneyepithelial cell line (CVI/EBNA) based on its mitogenic activity on themurine T cell line, CTLL-2. IL-15 was also independently discovered as acytokine produced by a human adult T cell leukemia cell line (HuT-102)that stimulated T cell proliferation and was designated IL-T. By virtueof its activity as a stimulator of T cells, NK cells, LAK cells, andTILs, IL-2 is currently in clinical trials testing its potential use intreatments for cancer and for viral infections. Because of its similarbiological activities, IL-15 should have similar therapeutic potential.

Chemokines

Chemokines are a superfamily of mostly small, secreted proteins thatfunction in leukocyte trafficking, recruiting, and recirculation. Theyalso play a critical role in many pathophysiological processes such asallergic responses, infectious and autoimmune diseases, angiogenesis,inflammation, tumor growth, and hematopoietic development. Approximately80 percent of these proteins have from 66 to 78 amino acids (aa) intheir mature form. The remainder are larger with additional aa occurringupstream of the protein core or as part of an extended C-terminalsegment. All chemokines signal through seven transmembrane domainG-protein coupled receptors. There are at least seventeen knownchemokine receptors, and many of these receptors exhibit promiscuousbinding properties whereby several different chemokines can signalthrough the same receptor.

Chemokines are divided into subfamilies based on conserved aa sequencemotifs. Most family members have at least four conserved cysteineresidues that form two intramolecular disulfide bonds. The subfamiliesare defined by the position of the first two cysteine residues:

-   -   The alpha subfamily, also called the CXC chemokines, have one aa        separating the first two cysteine residues. This group can be        further subdivided based on the presence or absence of a        glu-leu-arg (ELR) aa motif immediately preceding the first        cysteine residue. There are currently five CXC-specific        receptors and they are designated CXCR1 to CXCR5. The ELR⁺        chemokines bind to CXCR2 and generally act as neutrophil        chemoattractants and activators. The ELR− chemokines bind CXCR3        to −5 and act primarily on lymphocytes. At the time of this        writing, 14 different human genes encoding CXC chemokines have        been reported in the scientific literature with some additional        diversity contributed by alternative splicing.    -   In the beta subfamily, also called the CC chemokines, the first        two cysteines are adjacent to one another with no intervening        aa. There are currently 24 distinct human beta subfamily        members. The receptors for this group are designated CCR1 to        CCR11. Target cells for different CC family members include most        types of leukocytes.    -   There are two known proteins with chemokine homology that fall        outside of the alpha and beta subfamilies. Lymphotactin is the        lone member of the gamma class (C chemokine) which has lost the        first and third cysteines. The lymphotactin receptor is        designated XCR1. Fractalkine, the only known member of the delta        class (CX₃C chemokine), has three intervening aa between the        first two cysteine residues. This molecule is unique among        chemokines in that it is a transmembrane protein with the        N-terminal chemokine domain fused to a long mucin-like stalk.        The fractalkine receptor is known as CX₃CR1.        VEGF

The present invention is also applicable to Vasculature EndothelialGrowth Factor (VEGF). Angiogenesis is a process of new blood vesseldevelopment from pre-existing vasculature. It plays an essential role inembryonic development, normal growth of tissues, wound healing, thefemale reproductive cycle (i.e., ovulation, menstruation and placentaldevelopment), as well as a major role in many diseases. Particularinterest has focused on cancer, since tumors cannot grow beyond a fewmillimeters in size without developing a new blood supply. Angiogenesisis also necessary for the spread and growth of tumor cell metastases.

One of the most important growth and survival factors for endothelium isVEGF. VEGF induces angiogenesis and endothelial cell proliferation andit plays an important role in regulating vasculogenesis. VEGF is aheparin-binding glycoprotein that is secreted as a homodimer of 45 kDa.Most types of cells, but usually not endothelial cells themselves,secrete VEGF. Since the initially discovered VEGF, VEGF-A, increasesvascular permeability, it was known as vascular permeability factor. Inaddition, VEGF causes vasodilatation, partly through stimulation ofnitric oxide synthase in endothelial cells. VEGF can also stimulate cellmigration and inhibit apoptosis. There are several splice variants ofVEGF-A. The major ones include: 121, 165, 189 and 206 amino acids (aa),each one comprising a specific exon addition.

EMAP II

Endothelial-Monocyte Activating Polypeptide-II (EMAP-II) is a cytokinethat is an antiangiogenic factor in tumor vascular development, andstrongly inhibits tumor growth. Recombinant human EMAP-II is an 18.3 kDaprotein containing 166 amino acid residues. EMAP II has also bee foundto increase endothelial vessel permeability.

PDGF

It has also been proposed that platelet-derived growth factor (PDGF)antagonists might increase drug-uptake and therapeutic effects of abroad range of anti-tumor agents in common solid tumors. PDGF is acytokine of 30 kDA and is released by platelets on wounding andstimulates nearby cells to grow and repair the wound.

PD-ECGF

As its name suggests, platelet-derived endothelial cell growth factor(PD-ECGF) was originally isolated from platelets based on its ability toinduce mitosis in endothelial cells. Its related protein is gliostatin.

Targeting Moiety

We have found that the therapeutic index of cytokines can be increasedby homing of targeting the cytokine to tumor vessels. In addition, sinceit is known that tumor cells can form part of the lining of tumorvasculature, the present invention encompasses targeting to tumor cellsdirectly as well as to its vasculature. Any convenient tumor or tumorvasculature, particular endothelial cell, targeting moiety may be usedin the conjugate of the present invention. Many such targeting moietiesare known and these and any which subsequently become available areencompassed within the scope of the present invention. In oneembodiment, the targeting moiety is a binding partner, such as a ligand,of a receptor expressed by a tumor cell, or a binding partner, such asan antibody, to a marker or a component of the extracellular matrixassociated with tumor cells. More particularly the targeting moiety isbinding partner, such as a ligand of, a receptor expressed bytumor-associated vessels, or a binding partner, such as an antibody, toan endothelial marker or a component of the extracellular matrixassociated with angiogenic vessels. The term binding partner is usedhere in its broadest sense and includes both natural and syntheticbinding domains, including ligand and antibodies or binding fragmentsthereof. Thus, said binding partner can be an antibody or a fragmentthereof such as Fab, Fv, single-chain Fv, a peptide or apeptido-mimetic, namely a peptido-like molecule capable of binding tothe receptor, marker of extracellular component of the cell.

The following represent a non-limiting examples of suitable targetingdomains and receptors/markers to which the conjugate may be targeted:

CD13

It has surprisingly been found that the therapeutic index of certaincytokines can be remarkably improved and their immunotherapeuticproperties can be enhanced by coupling with a ligand of aminopeptidase-Nreceptor (CD13). CD13 is a trans-membrane glycoprotein of 150 kDa highlyconserved in various species. It is expressed on normal cells as well asin myeloid tumor lines, in the angiogenic endothelium and is someepithelia. CD113 receptor is usually identified as “NGR” receptor, inthat its peptide ligands share the amino acidic “NGR” motif The ligandis preferably a straight or cyclic peptide comprising the NGR motif,such as CNGRCVSGCAGRC, NGRAHA, GNGRG, cycloCVLNGRMEC or cycloCNGRC, ormore preferably the peptide CNGRC. Further details can be found in ourWO01/61017 which is incorporated herein by reference.

TNF Receptor

As with members of the TNF Superfamily, members of the TNF ReceptorSuperfamily (TNFRSF) also share a number of common features. Inparticular, molecules in the TNFRSF are all type I (N-terminusextracellular) transmembrane glycoproteins that contain one to sixligand-binding, 40 aa residue cysteine-rich motifs in theirextracellular domain. In addition, functional TNFRSF members are usuallytrimeric or multimeric complexes that are stabilised by intracysteinedisulfide bonds. Unlike most members of the TNFSF, TNFRSF members existin both membrane-bound and soluble forms. Finally, although aa sequencehomology in the cytoplasmic domains of the superfamily members does notexceed 25%, a number of receptors are able to transduce apoptoticsignals in a variety of cells, suggesting a common function.

CD40: CD40 is a 50 kDa, 277 aa residue transmembrane glycoprotein mostoften associated with B cell proliferation and differentiation.Expressed on a variety of cell types, human CD40 cDNA encodes a 20 aaresidue signal sequence, a 173 aa residue extracellular region, a 22 aaresidue transmembrane segment, and a 62 aa residue cytoplasmic domain.There are four cysteine-rich motifs in the extracellular region that areaccompanied by a juxtamembrane sequence rich in serines and threonines.Cells known to express CD40 include endothelial cells.

TNFRI/p55/CD120a: TNFRI is a 55 kDa, 455 aa residue transmembraneglycoprotein that is apparently expressed by virtually all nucleatedmammalian cells. The molecule has a 190 aa residue extracellular region,a 25 aa residue transmembrane segment, and a 220 aa residue cytoplasmicdomain. Both TNF-α and TNF-β bind to TNFRI. Among the numerous cellsknown to express TNFRI are endothelial cells.

TNFRII/p75/CD120b: Human TNFRII is a 75 kDa, 461 aa residuetransmembrane glycoprotein originally isolated from a human lungfibroblast library. This receptor consists of a 240 aa residueextracellular region, a 27 aa residue transmembrane segment and a 173 aaresidue cytoplasmic domain. Soluble forms of TNFRII have beenidentified, resulting apparently from proteolytic cleavage by ametalloproteinase termed TRRE (TNF-Receptor Releasing Enzyme). Theshedding process appears to be independent of that for soluble TNFRI.Among the multitude of cells known to express TNFRII are endothelialcells.

CD134L/OX40L: OX40, the receptor for OX40L, is a T cell activationmarker with limited expression that seems to promote the survival (andperhaps prolong the immune response) of CD4⁺ T cells at sites ofinflammation. OX40L also shows limited expression. Currently onlyactivated CD4⁺, CD8⁺ T cells, B cells, and vascular endothelial cellshave been reported to express this factor. The human ligand is a 32 kDa,183 aa residue glycosylated polypeptide that consists of a 21 aa residuecytoplasmic domain, a 23 aa residue transmembrane segment, and a 139 aaresidue extracellular region.

VEGF Receptor Family

There are three receptors in the VEGF receptor family. They have thecommon properties of multiple IgG-like extracellular domains andtyrosine kinase activity. The enzyme domains of VEGF receptor 1 (VEGFR1, also known as Flt-1), VEGF R2 (also known as KDR or Flk-1), and VEGFR3 (also known as Flt-4) are divided by an inserted sequence.Endothelial cells also express additional VEGF receptors, Neuropilin-1and Neuropilin-2. VEGF-A binds to VEGF R1 and VEGF R2 and toNeuropilin-1 and Neuropilin-2. P1GF and VEGF-B bind VEGF R1 andNeuropilin-1. VEGF-C and -D bind VEGF R3 and VEGF R2. HIV-tat andpeptides derived therefrom have also been found to target the VEGFR.

PDGF Receptors

PDGF receptors are expressed in the stromal compartment in most commonsolid tumors. Inhibition of stromally expressed PDGF receptors in a ratcolon carcinoma model reduces the tumor interstitial fluid pressure andincreases tumor transcapillary transport.

PSMA

Prostate specific membrane antigen (PSMA) is also an excellent tumorendothelial marker, and PSMA antibodies can be generated.

Cell Adhesion Molecules (CAMs)

Cell adhesion molecules (CAMs) are cell surface proteins involved in thebinding of cells, usually leukocytes, to each other, to endothelialcells, or to extracellular matrix. Specific signals produced in responseto wounding and infection control the expression and activation ofcertain of these adhesion molecules. The interactions and responses theninitiated by binding of these CAMs to their receptors/ligands playimportant roles in the mediation of the inflammatory and immunereactions that constitute one line of the body's defence against theseinsults. Most of the CAMs characterised so far fall into three generalfamilies of proteins: the immunoglobulin (Ig) superfamily, the integrinfamily, or the selectin family.

A member of the Selectin family of cell surface molecules, L-Selectinconsists of an NH2-terminal lectin type C domain, an EGF-like domain,two complement control domains, a 15 amino acid residue spacer, atransmembrane sequence and a short cytoplasmic domain.

Three ligands for L-Selectin on endothelial cells have been identified,all containing O-glycosylated mucin or mucin-like domains. The firstligand, GlyCAM-1, is expressed almost exclusively in peripheral andmesenteric lymph node high endothelial venules. The second L-Selectinligand, originally called sgp90, has now been shown to be CD34. Thissialomucin-like glycoprotein, often used as a surface marker for thepurification of pluripotent stem cells, shows vascular expression in awide variety of nonlymphoid tissues, as well as on the capillaries ofperipheral lymph nodes. The third ligand for L-Selectin is MadCAM 1, amucin-like glycoprotein found on mucosal lymph node high endothelialvenules.

P-Selectin, a member of the Selectin family of cell surface molecules,consists of an NH2-terminal lectin type C domain, an EGF-like domain,nine complement control domains, a transmembrane domain, and a shortcytoplasmic domain.

The tetrasaccharide sialyl Lewisx (sLex) has been identified as a ligandfor both P- and E-Selectin, but P- E- and L-Selectin can all bind sLexand sLea under appropriate conditions. P-Selectin also reportedly bindsselectively to a 160 kDa glycoprotein present on murine myeloid cellsand to a glycoprotein on myeloid cells, blood neutrophils, monocytes,and lymphocytes termed P-Selectin glycoprotein ligand-1 (PSGL-1), aligand that also can bind E-Selectin. P-Selectin-mediated rolling ofleukocytes can be completely inhibited by a monoclonal antibody specificfor PSLG-1, suggesting that even though P-Selectin can bind to a varietyof glycoproteins under in vitro conditions, it is likely thatphysiologically important binding is more limited. A variety of evidenceindicates that P-Selectin is involved in the adhesion of myeloid cells,as well as B and a subset of T cells, to activated endothelium.

Ig Superfamily CAMs

The Ig superfamily CAMs are calcium-independent transmembraneglycoproteins. Members of the Ig superfamily include the intercellularadhesion molecules (ICAMs), vascular-cell adhesion molecule (VCAM-1),platelet-endothelial-cell adhesion molecule (PECAM-1), and neural-celladhesion molecule (NCAM). Each Ig superfamily CAM has an extracellulardomain, which contains several Ig-like intrachain disulfide-bonded loopswith conserved cysteine residues, a transmembrane domain, and anintracellular domain that interacts with the cytoskeleton. Typically,they bind integrins or other Ig superfamily CAMs. The neuronal CAMs havebeen implicated in neuronal patterning. Endothelial CAMs play animportant role in immune response and inflammation.

In more detail, vascular cell adhesion molecule (VCAM-1, CD106, orINCAM-110), platelet endothelial cell adhesion molecule (PECAM-I/CD31)and intercellular adhesion molecules 1, 2 &3 (ICAM-1, 2 & 3) are fivefunctionally related CAM/IgSF molecules that are critically involved inleukocyte-connective tissue/endothelial cell interactions. Expressedprincipally on endothelial cells, these molecules in general regulateleukocyte migration across blood vessel walls and provide attachmentpoints for developing endothelium during angiogenesis and are allsuitable for targeting in the present invention.

Human CD31 is a 130 kDa, type I (extracellular N-terminus) transmembraneglycoprotein that belongs to the cell adhesion molecule (CAM) or C2-likesubgroup of the IgSFl. The mature molecule is 711 amino acid (aa)residues in length and contains a 574 aa residue extracellular region, a19 aa residue transmembrane segment, and a 118 aa residue cytoplasmictail. In the extracellular region, there are nine potential N-linkedglycosylation sites, and, with a predicted molecular weight of 80 kDa,it appears many of these sites are occupied. The most striking featureof the extracellular region is the presence of six Ig-homology unitsthat resemble the C2 domains of the IgSF. Although they vary in number,the presence of these modules is a common feature of all IgSF adhesionmolecules (ICAM-1, 2, 3 & VCAM-1).

Integrins

Integrins are non-covalently linked heterodimers of α and β subunits. Todate, 16 α subunits and 8 β subunits have been identified. These cancombine in various ways to form different types of integrin receptors.The ligands for several of the integrins are adhesive extracellularmatrix (ECM) proteins such as fibronectin, vitronectin, collagens andlaminin. Many integrins recognise the amino acid sequence RGD(arginine-glycine-aspartic acid) which is present in fibronectin or theother adhesive proteins to which they bind. Peptides and proteinfragments containing the RGD sequence can be used to modulate theactivities of the RGD-recognising integrins. Thus the present inventionmay employ as the targeting moiety peptides recognised by integrins.These peptides are conventionally known as “RGD-containing peptides”.These peptides may include peptides motifs which have been identified asbinding to integrins. These motifs include the amino acid sequences:DGR, NGR and CRGDC. The peptide motifs may be linear or cyclic. Suchmotifs are described in more detail in the following patents which areherein incorporated by reference in relation to their description of anRGD peptides: U.S. Pat. No. 5,536,814 which describes cyclasized CRGDCL,CRGDCA and GACRGDCLGA. U.S. Pat. No. 4,578,079 relates to syntheticpeptides of formula X-RGD-T/C—Y where X and Y are amino acids. U.S. Pat.No. 5,547,936 describes a peptide counting the sequence X-RGD-XX where Xmay be an amino acid. U.S. Pat. No. 4,988,621 describes a number ofRGD-counting peptides. U.S. Pat. No. 4,879,237 describes a generalpeptide of the formula RGD-Y where Y is an amino acid, and the peptideG-RGD-AP. U.S. Pat. No. 5,169,930 describes the peptide RGDSPK whichbinds to αvβ1 integrin. U.S. Pat. Nos. 5,498,694 and 5,700,908 relate tothe cytoplasmic domain of the β3 integrin sub-unit that strictlyspeaking is not an RGD-containing peptide; although it does contain thesequence RDG. WO97/08203 describes cyclic peptides that are structuralmimics or RGD-binding sites. U.S. Pat. No. 5,612,311 describes 15RGD-containing peptides that are capable of being cyclized either by C-Clinkage or through other groups such as penicillamine or mecaptopropionic acid analogs. U.S. Pat. No. 5,672,585 describes a generalformula encompassing RGD-containing peptides. A preferred group ofpeptides are those where the aspartic acid residue of RGD is derivatisedinto an O-methoxy tyrosine derivative. U.S. Pat. No. 5,120,829 describesan RGD cell attachment promoting binding site and a hydrophobicattachment domain. The D form is described in U.S. Pat. No. 5,587,456.U.S. Pat. No. 5,648,330 describes a cyclic RGD-containing peptide thathas high affinity for GP Iib/IIIa.

In a preferred embodiment of the present invention the targeting moietyis a ligand for αv β3 or αv β5 integrin.

The use of alpha v beta 3 ligands to convey cytotoxic chemotherapeuticdrugs to tumors has been previously reported (WPI 99-215158/199918.).However, in these patent application the idea was to deliver to tumorvessels toxic compounds, such as chemotherapeutic drugs or toxins oranti-angiogenic compounds.

In sharp contrast, TNF is an activator of endothelial and immune cellfunctions, rather than an inhibitor or a toxic compound. For instanceTNF is believed to be a pro-angiogenic molecule and not ananti-angiogenic molecule. Moreover, despite TNF was discovered for itscytotoxicity against some tumor cell lines, it is well known that TNFcan seldom kill cells in culture, if protective mechanisms are notblocked, (e.g. with transcription/translation inhibitors).

It would appear therefore that the anti-tumor activity of TNF is basedon its activating effects on various cells, and little or not to directcytotoxic effects on tumor cells or endothelial cells. TNF should beviewed in this context as a biological response modifier and not as aclassical cytotoxic compound.

Thus, the therapeutic properties of TNF delivered to alpha v beta 3 arenot obvious, simply on the bases of the disclosure of patent WPI99-215158/199918.

Molecules containing the ACDCRGDCFCG motif are expected to targetactivated murine as well human vessels (72). Thus, one may expect thathuman RGD-TNF is endowed with better anti-tumor properties than humanTNF in patients, as we observed with the murine counterparts in mice.

The maximum tolerated dose of bolus TNF (intravenous) in humans is218-410 μg/m² (28), about 10-fold lower than the effective dose inanimals (29). Based on data from murine models it is believed that 10-50times higher dose is necessary to achieve anti-tumor effects in humans(35). In the first clinical study on hyperthermic isolated-limbperfusion, high response rates were obtained with the unique dose of 4mg of TNF in combination with melphalan and interferon-γ (32). Otherworks showed that interferon-γ can be omitted and that even lower dosesof TNF can be sufficient to induce a therapeutic response (33, (34).Since also these treatments are not devoid of risk of toxicity (35), theuse of RGD-TNF may represent an alternative approach to reduce toxiceffects at least in this setting.

Moreover, the RGD-TNF cDNA could be used for gene therapy purposes inplace of the TNF gene (76) whereas biotinylated RGD-TNF could beapplied, in principle, in combination tumor pre-targeting withbiotinylated antibodies and avidin (71), to further increase itstherapeutic index.

Activin

Cells known to express ActRII include endothelial cells. ActRIIBexpression parallels that for ActRII, and is again found in endothelialcells. Cells known to express ActRI include vascular endothelial cells.ActRIB has also been identified in endothelial cells.

Angiogenin

Angiogenin (ANG) is a 14 kDa, non-glycosylated polypeptide so named forits ability to induce new blood vessel growth.

Annexin V

Annexin V is a member of a calcium and phospholipid binding family ofproteins with vascular anticoagulant activity. Various synomyms forAnnexin V exist: placental protein 4 (PP4), placental anticoagulantprotein I (PAP I), calphobindin I (CPB-I), calcium dependentphospholipid binding protein 33 (CaBP33), vascular anticoagulant proteinalpha (VACa), anchorin CII, lipocortin-V, endonexin II, andthromboplastin inhibitor. The number of binding sites for Annexin V hasbeen reported as 6-24×106/cell in tumor cells and 8.8×106/cell forendothelial cells.

CD44

Another molecule apparently involved in white cell adhesive events isCD44, a molecule ubiquitously expressed on both hematopoietic andnon-hematopoietic cells. CD44 is remarkable for its ability to generatealternatively spliced forms, many of which differ in their activities.This remarkable flexibility has led to speculation that CD44, via itschanging nature, plays a role in some of the methods that tumor cellsuse to progress successfully through growth and metastasis. CD44 is a80-250 kDa type I (extracellular N-terminus) transmembrane glycoprotein.Cells known to express CD44H include vascular endothelial cells.

There are multiple ligands for CD44, including osteopontin, fibronectin,collagen types I and IV and hyaluronate. Binding to fibronectin isreported to be limited to CD44 variants expressing chrondroitin sulfate,with the chrondroitin sulfate attachment site localised to exons v8-v11.Hyaluronate binding is suggested to be possible for virtually all CD44isoforms. One of the principal binding sites is proposed to be centredin exon 2 and to involve lysine and arginine residues. Factors otherthan the simple expression of a known hyaluronate-binding motif alsoappear to be necessary for hyaluronate binding. Successful hyaluronatebinding is facilitated by the combination of exons expressed, adistinctive cytoplasmic tail, glycosylation patterns, and the activitystate of the cell. Thus, in terms of its hyaluronate-binding function, agreat deal of “potential” flexibility exists within each CD44-expressingcell.

Fibroblast Growth Factor (FGF)

The name “fibroblast growth factor” (FGF) is a limiting description forthis family of cytokines. The function of FGFs is not restricted to cellgrowth. Although some of the FGFs do, indeed, induce fibroblastproliferation, the original FGF molecule (FGF-2 or FGF basic) is nowknown to also induce proliferation of endothelial cells, chondrocytes,smooth muscle cells, melanocytes, as well as other cells. It can alsopromote adipocyte differentiation, induce macrophage and fibroblast IL-6production, stimulate astrocyte migration, and prolong neuronalsurvival. To date, the FGF superfamily consists of 23 members, all ofwhich contain a conserved 120 amino acid (aa) core region that containssix identical, interspersed amino acids.

FGF-1: Human FGF-1 (also known as FGF acidic, FGFa, ECGF and HBGF-1) isa 17-18 kDa non-glycosylated polypeptide that is expressed by a varietyof cells from all three germ layers. The binding molecule may be eitheran FGF receptor. Cells known to express FGF-1 include endothelial cells.

FGF-2: Human FGF-2, otherwise known as FGF basic, HBGF-2, and EDGF, isan 18 kDa, non-glycosylated polypeptide that shows both intracellularand extracellular activity. Following secretion, FGF-2 is sequestered oneither cell surface HS or matrix glycosaminoglycans. Although FGF-2 issecreted as a monomer, cell surface HS seems to dimerize monomeric FGF-2in a non-covalent side-to-side configuration that is subsequentlycapable of dimerizing and activating FGF receptors. Cells known toexpress FGF-2 include endothelial cells.

FGF-3: Human FGF-3 is the product of the int-2 gene [i.e., derived fromintegration region-2, a region on mouse chromosome 7 that contains agene (int-2/FGF-3) accidentally activated following retroviralinsertion]. The molecule is synthesised as a 28-32 kDa, 222 aaglycoprotein that contains a number of peptide motifs. Cells reported toexpress FGF-3 are limited to developmental cells and tumors. Tumorsknown to express FGF-3 include breast carcinomas and colon cancer celllines.

FGF-4: Human FGF-4 is a 22 kDa, 176 aa glycoprotein that is the productof a developmentally-regulated gene. The molecule is synthesised as a206 aa precursor that contains a large, ill-defined 30 aa signalsequence plus two heparin-binding motifs (at aa 51-55 and 140-143). Theheparin-binding sites directly relate to FGF-4 activity; heparin/heparanregulate the ability of FGF-4 to activate FGFR1 and FGFR2. Cells knownto express FGF-4 include both tumor cells and embryonic cells. Itsidentification in human stomach cancer gives rise to one alternativedesignation (/hst-1/hst), while its isolation in Kaposi's sarcomaprovides grounds for another (K-FGF).

IL-1R

IL-1 exerts its effects by binding to specific receptors. Two distinctIL-1 receptor binding proteins, plus a non-binding signalling accessoryprotein have been identified. Each have three extracellularimmunoglobulin-like (Ig-like) domains, qualifying them for membership inthe type IV cytokine receptor family. The two receptor binding proteinsare termed type I IL-1 receptor (IL-1 RI) and type II IL-1 receptor(IL-1 RII) respectively. Human IL-1 R1 is a 552 aa, 80 kDa transmembraneglycoprotein that has been isolated from endothelium cells.

RTK

The new family of receptor tyrosine kinase (RTK), the Eph receptors andtheir ligands ephrins, have been found to be involved in vascularassembly, angiogenesis, tumorigenesis, and metastasis. It has also beenthat class A Eph receptors and their ligands are elevated in tumor andassociated vasculature.

MMP

Matrix metalloproteinases (MMPs) have been implicated in tumor growth,angiogenesis, invasion, and metastasis. They have also been suggestedfor use as tumor markers.

NG2

NG2 is a large, integral membrane, chondroitin sulfate proteoglycan thatwas first identified as a cell surface molecule expressed by immatureneural cells. Subsequently NG2 was found to be expressed by a widevariety of immature cells as well as several types of tumors with highmalignancy. NG2 has been suggested as a target molecule in the tumorvasculature. In particular, collagenase-1 (C1) is the predominant matrixmetalloproteinase present in newly formed microvessels and serves as amarker of neovascularization.

Oncofetal Fibronectin

The expression of the oncofetal fragment of fibronectin (Fn-f) has alsobeen found to be increased during angiogenesis and has been suggested asa marker of tumor angiogenesis. In one embodiment the TTM is an antibodyor fragment thereof to the oncofetal ED-B domain of fibronectin. Thepreparation of such an antibody and its conjugation with IL-12 isdescribed in Halin et al (2002) Nature Biotechnology 20:264-269.

Tenascin

Tenascin is a matrix glycoprotein seen in malignant tumors includingbrain and breast cancers and melanoma. Its expression is malignant butnot well differentiated tumors and association with the blood vessels oftumors makes it an important target for both understanding the biologyof malignant tumors and angiogenesis, but is a therapeutic cancer targetand marker as well.

The targeting moiety is preferably a polypeptide which is capable ofbinding to a tumor cell or tumor vasculature surface molecule. As wellas those mentioned above other such surface molecules which are known orbecome available may also be targeted by the first sequence.

It will be appreciated that one can apply conventional protein bindingassays to identify molecules which bind to surface molecules. It willalso be appreciated that one can apply structural-based drug design todevelop sequences which bind to surface molecules.

High throughput screening, as described above for synthetic compounds,can also be used for identifying targeting molecules.

This invention also contemplates the use of competitive drug screeningassays in which neutralising antibodies capable of binding a targetspecifically compete with a test compound for binding to a target.

Binding Partner (BP)

The targeting moiety generally take the form of a binding partner (BP)to a surface molecule comprising or consisting of one or more bindingdomains.

Ligand

The targeting moiety of the present invention may take the form of aligand. The ligands may be natural or synthetic. The term “ligand” alsorefers to a chemically modified ligand. The one or more binding domainsof the BP may consist of, for example, a natural ligand for a receptor,which natural ligand may be an adhesion molecule or a growth-factorreceptor ligand (e.g. epidermal growth factor), or a fragment of anatural ligand which retains binding affinity for the receptor.

Synthetic ligands include the designer ligands. As used herein, the termmeans “designer ligands” refers to agents which are likely to bind tothe receptor based on their three dimensional shape compared to that ofthe receptor.

Antibodies

Alternatively, the binding domains may be derived from heavy and lightchain sequences from an immunoglobulin (Ig) variable region. Such avariable region may be derived from a natural human antibody or anantibody from another species such as a rodent antibody. Alternativelythe variable region may be derived from an engineered antibody such as ahumanised antibody or from a phage display library from an immunised ora non-immunised animal or a mutagenised phage-display library. As asecond alternative, the variable region may be derived from asingle-chain variable fragment (scFv). The BP may contain othersequences to achieve multimerisation or to act as spacers between thebinding domains or which result from the insertion of restriction sitesin the genes encoding the BP, including Ig hinge sequences or novelspacers and engineered linker sequences.

The BP may comprise, in addition to one or more immunoglobulin variableregions, all or part of an Ig heavy chain constant region and so maycomprise a natural whole Ig, an engineered Ig, an engineered Ig-likemolecule, a single-chain Ig or a single-chain Ig-like molecule.Alternatively, or in addition, the BP may contain one or more domainsfrom another protein such as a toxin.

As used herein, an “antibody” refers to a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genes orfragments of immunoglobulin genes. Antibodies may exist as intactimmunoglobulins or as a number of fragments, including thosewell-characterised fragments produced by digestion with variouspeptidases. While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate thatantibody fragments may be synthesised de novo either chemically or byutilising recombinant DNA methodology. Thus, the term antibody, as usedherein also includes antibody fragments either produced by themodification of whole antibodies or synthesised de novo usingrecombinant DNA methodologies. Antibody fragments encompassed by the useof the term “antibodies” include, but are not limited to, Fab, Fab′,F(ab′)2, scFv, Fv, dsFv diabody, and Fd fragments.

The invention also provides monoclonal or polyclonal antibodies to thesurface proteins. Thus, the present invention further provides a processfor the production of monoclonal or polyclonal antibodies topolypeptides of the invention.

If polyclonal antibodies are desired, a selected mammal (e.g., mouse,rabbit, goat, horse, etc.) is immunised with an immunogenic polypeptidebearing an epitope(s). Serum from the immunised animal is collected andtreated according to known procedures. If serum containing polyclonalantibodies to an epitope contains antibodies to other antigens, thepolyclonal antibodies can be purified by immunoaffinity chromatography.Techniques for producing and processing polyclonal antisera are known inthe art. In order that such antibodies may be made, the invention alsoprovides polypeptides of the invention or fragments thereof haptenisedto another polypeptide for use as immunogens in animals or humans.

Monoclonal antibodies directed against binding cell surface epitopes inthe polypeptides can also be readily produced by one skilled in the art.The general methodology for making monoclonal antibodies by hybridomasis well known. Immortal antibody-producing cell lines can be created bycell fusion, and also by other techniques such as direct transformationof B lymphocytes with oncogenic DNA, or transfection with Epstein-Barrvirus. Panels of monoclonal antibodies produced against epitopes can bescreened for various properties; i.e., for isotype and epitope affinity.

An alternative technique involves screening phage display librarieswhere, for example the phage express scFv fragments on the surface oftheir coat with a large variety of complementarity determining regions(CDRs). This technique is well known in the art.

For the purposes of this invention, the term “antibody”, unlessspecified to the contrary, includes fragments of whole antibodies whichretain their binding activity for a target antigen. As mentioned abovesuch fragments include Fv, F(ab′) and F(ab′)₂ fragments, as well assingle chain antibodies (scFv). Furthermore, the antibodies andfragments thereof may be humanised antibodies, for example as describedin EP-A-239400.

Screens

In one aspect, the invention relates to a method of screening for anagent capable of binding to a tumor or tumor vasculature cell surfacemolecule, the method comprising contacting the cell surface moleculewith an agent and determining if said agent binds to said cell surfacemolecule.

As used herein, the term “agent” includes, but is not limited to, acompound, such as a test compound, which may be obtainable from orproduced by any suitable source, whether natural or not. The agent maybe designed or obtained from a library of compounds which may comprisepeptides, as well as other compounds, such as small organic moleculesand particularly new lead compounds. By way of example, the agent may bea natural substance, a biological macromolecule, or an extract made frombiological materials such as bacteria, fungi, or animal particularlymammalian) cells or tissues, an organic or an inorganic molecule, asynthetic test compound, a semi-synthetic test compound, a structural orfunctional mimetic, a peptide, a peptidomimetics, a derivatised testcompound, a peptide cleaved from a whole protein, or a peptidessynthesised synthetically (such as, by way of example, either using apeptide synthesizer) or by recombinant techniques or combinationsthereof, a recombinant test compound, a natural or a non-natural testcompound, a fusion protein or equivalent thereof and mutants,derivatives or combinations thereof.

The agent can be an amino acid sequence or a chemical derivativethereof. The substance may even be an organic compound or otherchemical. The agent may even be a nucleotide sequence—which may be asense sequence or an anti-sense sequence.

Protein

The term “protein” includes single-chain polypeptide molecules as wellas multiple-polypeptide complexes where individual constituentpolypeptides are linked by covalent or non-covalent means. The term“polypeptide” includes peptides of two or more amino acids in length,typically having more than 5, 10 or 20 amino acids.

Polypeptide Homologues

It will be understood that polypeptide sequences for use in theinvention are not limited to the particular sequences or fragmentsthereof but also include homologous sequences obtained from any source,for example related viral/bacterial proteins, cellular homologues andsynthetic peptides, as well as variants or derivatives thereof.Polypeptide sequences of the present invention also include polypeptidesencoded by polynucleotides of the present invention.

Polypeptide Variants, Derivatives and Fragments

The terms “variant” or “derivative” in relation to the amino acidsequences of the present invention includes any substitution of,variation of, modification of, replacement of, deletion of or additionof one (or more) amino acids from or to the sequence providing theresultant amino acid sequence preferably has targeting activity,preferably having at least 25 to 50% of the activity as the polypeptidespresented in the sequence listings, more preferably at leastsubstantially the same activity.

Thus, sequences may be modified for use in the present invention.Typically, modifications are made that maintain the activity of thesequence. Thus, in one embodiment, amino acid substitutions may be made,for example from 1, 2 or 3 to 10, 20 or 30 substitutions provided thatthe modified sequence retains at least about 25 to 50% of, orsubstantially the same activity. However, in an alternative embodiment,modifications to the amino acid sequences of a polypeptide of theinvention may be made intentionally to reduce the biological activity ofthe polypeptide. For example truncated polypeptides that remain capableof binding to target molecule but lack functional effector domains maybe useful.

In general, preferably less than 20%, 10% or 5% of the amino acidresidues of a variant or derivative are altered as compared with thecorresponding region depicted in the sequence listings.

Amino acid substitutions may include the use of non-naturally occurringanalogues, for example to increase blood plasma half-life of atherapeutically administered polypeptide (see below for further detailson the production of peptide derivatives for use in therapy).Conservative substitutions may be made, for example according to theTable below. Amino acids in the same block in the second column andpreferably in the same line in the third column may be substituted foreach other: ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M NQ Polar - charged D E K R AROMATIC H F W Y

Polypeptides of the invention also include fragments of the abovementioned polypeptides and variants thereof, including fragments of thesequences. Preferred fragments include those which include an epitope.Suitable fragments will be at least about 5, e.g. 10, 12, 15 or 20 aminoacids in length. They may also be less than 200, 100 or 50 amino acidsin length. Polypeptide fragments of the proteins and allelic and speciesvariants thereof may contain one or more (e.g. 2, 3, 5, or 10)substitutions, deletions or insertions, including conservedsubstitutions. Where substitutions, deletion and/or insertions have beenmade, for example by means of recombinant technology, preferably lessthan 20%, 10% or 5% of the amino acid residues depicted in the sequencelistings are altered.

Proteins of the invention are typically made by recombinant means, forexample as described below. However they may also be made by syntheticmeans using techniques well known to skilled persons such as solid phasesynthesis. Various techniques for chemical synthesising peptides arereviewed by Borgia and Fields, 2000, TibTech 18: 243-251 and describedin detail in the references contained therein.

Preparation

Methods for preparing CD13 L-IFN conjugates have been described inWO01/61017. For instance interferon gamma can be fused with the CNGRCpeptide by genetic engineering or by chemical synthesis. Given thedimeric structure of interferon gamma, conjugates bearing two CNGRCmoieties at the N-terminus or the C-terminus are preferable to providemultivalent high avidity interactions.

Using similar methods it is possible to prepare CRGDC-IFN-γ conjugatesto be used in combination with CNGRC-TNF.

It would be easy for a man skilled in the art to prepare conjugates ofalpha v beta 3-L-TNF with antibody or antibody fragments that targettumor cells, or tumor associated vessels to further increase the homingto tumor of this TNF derivatives. For instance, avb3L-TNF could becoupled with antibodies against tumor associates antigens or againstother tumor angiogenic markers, e.g. matrix metalloproteases (57) andvascular endothelial growth factor (58) or directed against componentsof the extracellular matrix, such as anti-tenascin antibodies oranti-fibronectin EDB domain.

The avb3L-TNF conjugate could be prepared in many ways. For instance theavb3L is an antibody or a fragment of it, preferably of human origin orbearing a humanized scaffold. In the preferred embody of the inventionthe avb3L is a peptide. For instance one peptide that bind to avb3 hasbeen recently discovered using phage-peptide libraries. This peptide ischaracterized by the presence of the sequence CRGDC. Peptides orantibodies can be coupled to TNF using well known recombinant DNAtechnologies or by chemical conjugation. These molecules could also beprepared by indirect conjugation: for instance they can be bothbiotinylated and coupled using tetravalent avidin as non covalentcross-linker.

Therapeutic Peptides

Peptides of the present invention may be administered therapeutically topatients. It is preferred to use peptides that do not consisting solelyof naturally-occurring amino acids but which have been modified, forexample to reduce immunogenicity, to increase circulatory half-life inthe body of the patient, to enhance bioavailability and/or to enhanceefficacy and/or specificity.

A number of approaches have been used to modify peptides for therapeuticapplication. One approach is to link the peptides or proteins to avariety of polymers, such as polyethylene glycol (PEG) and polypropyleneglycol (PPG)—see for example U.S. Pat. Nos. 5,091,176, 5,214,131 andU.S. Pat. No. 5,264,209.

Replacement of naturally-occurring amino acids with a variety of uncodedor modified amino acids such as D-amino acids and N-methyl amino acidsmay also be used to modify peptides.

Another approach is to use bifunctional crosslinkers, such asN-succinimidyl 3-(2 pyridyldithio)propionate, succinimidyl 6-[3-(2pyridyldithio)propionamido]hexanoate, and sulfosuccinimidyl 6-[3-(2pyridyldithio)propionamido]hexanoate (see U.S. Pat. No. 5,580,853).

It may be desirable to use derivatives of the peptides of the inventionwhich are conformationally constrained. Conformational constraint refersto the stability and preferred conformation of the three-dimensionalshape assumed by a peptide. Conformational constraints include localconstraints, involving restricting the conformational mobility of asingle residue in a peptide; regional constraints, involving restrictingthe conformational mobility of a group of residues, which residues mayform some secondary structural unit; and global constraints, involvingthe entire peptide structure.

The active conformation of the peptide may be stabilised by a covalentmodification, such as cyclization or by incorporation of gamma-lactam orother types of bridges. For example, side chains can be cyclized to thebackbone so as create a L-gamma-lactam moiety on each side of theinteraction site. See, generally, Hruby et al., “Applications ofSynthetic Peptides,” in Synthetic Peptides: A User's Guide: 259-345 (W.H. Freeman & Co. 1992). Cyclization also can be achieved, for example,by formation of cysteine bridges, coupling of amino and carboxy terminalgroups of respective terminal amino acids, or coupling of the aminogroup of a Lys residue or a related homolog with a carboxy group of Asp,Glu or a related homolog. Coupling of the alpha-amino group of apolypeptide with the epsilon-amino group of a lysine residue, usingiodoacetic anhydride, can be also undertaken. See Wood and Wetzel, 1992,Int'l J. Peptide Protein Res. 39: 533-39.

Another approach described in U.S. Pat. No. 5,891,418 is to include ametal-ion complexing backbone in the peptide structure. Typically, thepreferred metal-peptide backbone is based on the requisite number ofparticular coordinating groups required by the coordination sphere of agiven complexing metal ion. In general, most of the metal ions that mayprove useful have a coordination number of four to six. The nature ofthe coordinating groups in the peptide chain includes nitrogen atomswith amine, amide, imidazole, or guanidino functionalities; sulfur atomsof thiols or disulfides; and oxygen atoms of hydroxy, phenolic,carbonyl, or carboxyl functionalities. In addition, the peptide chain orindividual amino acids can be chemically altered to include acoordinating group, such as for example oxime, hydrazino, sulfhydryl,phosphate, cyano, pyridino, piperidino, or morpholino. The peptideconstruct can be either linear or cyclic, however a linear construct istypically preferred. One example of a small linear peptide isGly-Gly-Gly-Gly which has four nitrogens (an N₄ complexation system) inthe back bone that can complex to a metal ion with a coordination numberof four.

A further technique for improving the properties of therapeutic peptidesis to use non-peptide peptidomimetics. A wide variety of usefultechniques may be used to elucidating the precise structure of apeptide. These techniques include amino acid sequencing, x-raycrystallography, mass spectroscopy, nuclear magnetic resonancespectroscopy, computer-assisted molecular modelling, peptide mapping,and combinations thereof. Structural analysis of a peptide generallyprovides a large body of data which comprise the amino acid sequence ofthe peptide as well as the three-dimensional positioning of its atomiccomponents. From this information, non-peptide peptidomimetics may bedesigned that have the required chemical functionalities for therapeuticactivity but are more stable, for example less susceptible to biologicaldegradation. An example of this approach is provided in U.S. Pat. No.5,811,512.

Techniques for chemically synthesising therapeutic peptides of theinvention are described in the above references and also reviewed byBorgia and Fields, 2000, TibTech 18: 243-251 and described in detail inthe references contained therein.

Bifunctional Derivatives

A further embodiment of the invention is provided by bifunctionalderivatives in which the cytokines modified with a TTM are conjugatedwith antibodies, or their fragments, against tumoral antigens or othertumor angiogenic markers, e.g. αv integrins, metalloproteases or thevascular growth factor, or antibodies or fragments thereof directedagainst components of the extracellular matrix, such as anti-tenascinantibodies or anti-fibronectin EDB domain. The preparation of a fusionproduct between TNF and the hinge region of a mAb against thetumor-associated TAG72 antigen expressed by gastric and ovarianadenocarcinoma has recently been reported.

A further embodiment of the invention is provided by the tumoralpre-targeting with the biotin/avidin system. According to this approach,a ternary complex is obtained on the tumoral antigenic site, atdifferent stages, which is formed by 1) biotinylated mAb, 2) avidin (orstreptavidin) and 3) bivalent cytokine modified with the TTM and biotin.A number of papers proved that the pre-targeting approach, compared withconventional targeting with immunoconjugates, can actually increase theratio of active molecule homed at the target to free active molecule,thus reducing the treatment toxicity. This approach produced favorableresults with biotinylated TNF, which was capable of inducingcytotoxicity in vitro and decreasing the tumor cells growth underconditions in which normal TNF was inactive. The pre-targeting approachcan also be carried out with a two-phase procedure by using a bispecificantibody which at the same time binds the tumoral antigen and themodified cytokine. The use of a bispecific antibody directed against acarcinoembryonic antigen and TNF has recently been described as a meansfor TNF turmoral pre-targeting.

Tumour pre-targeting is another approach that as been recentlydeveloped. Pre-targeting can be performed with a variety of differentclasses of compounds according to a “two-step” or “three-step” approach(59). A specific example based on the avidin-biotin system applied tothe radioimmunoscintigraphy of tumours may be helpful in illustratingthe principle. In this case, a biotinylated mAb specific for atumour-associated antigen is administered first (the “targeting”molecule, first step). This is followed one day later by theadministration of avidin or streptavidin (the “chase” molecule, secondstep), tetravalent macromolecules that complex the biotinylated mAb andpromote the rapid removal of excess circulating molecules. Another daylater radionuclide-labeled biotin (the “effector” molecule, third step)is administered. This is at a time when both the “targeting” and “chase”macromolecules have been efficiently cleared from the circulation. Thisenables rapid diffusion and localization of the effector to the tumouras well as rapid excretion of excess, circulating free molecules. Thisis in clear contrast to directly labeled mAb which circulate forsignificantly longer periods of time thereby increasing backgrounds inradio-immunoscintigraphy and toxic side effects in radio-immunotherapy.Several reports have shown that the pre-targeting approach can indeedgreatly improve the target-to-blood ratio compared to conventionaltargeting with immuno-conjugates and decrease the toxicity of thetreatment (60, (61, (62, (63).

Application of the pre-targeting strategy to tumour therapy withbiotinylated TNF was considered to be of particular interest because ofthe markedly higher affinity of the biotin-avidin interaction (10⁻¹⁵M)compared to that of TNF-TNFR interactions. This was expected to allow anefficient, preferential binding of biotinylated TNF to pre-targetedcells over cells expressing TNFR and to prolong its persistence at thetumour site. On the basis of this rationale, the use of a three-stepmAb/avidin system for the targeting of biotinylated TNF has beenrecently described [Moro, 1997]. Mouse RMA lymphoma cells that had beentransfected with the Thy 1.1 allele to create a unique turn Gasparri etal 71). A similar approach could be exploited to further increase thetherapeutic index of biotinylated avb3L-TNF.

The avb3L-TNF pre-targeting strategy is not necessarily limited to a“three-step” approach. An example of a “two-step” approach, described inthe literature, is based on the use of a bispecific antibody with onearm specific for a tumour antigen and with the other for TNF. Inparticular, it has been recently described the use of a bispecificantibody directed against carcinoembryonic antigen and TNF to target TNFto tumours (64).

According to a further embodiment, the invention comprises a cytokineconjugated to both a TTM and an antibody, or a fragment thereof(directly or indirectly via a boitin-avidin bridge), on different TNFsubunits, where the antibody or its fragments are directed against anantigen expressed on tumor cells or other components of the tumorstroma, e.g. tenacin and fibronectin EDB domain. This results in afurther improvement of the tumor homing properties of the modifiedcytokine and in the slow release of the latter in the tumormicroenvironment through trimer-monomer-trimer transitions. The modifiedsubunits of e.g. TNF conjugates can disassociate from the targetingcomplexes and reassociate so as to form unmodified trimeric TNFmolecules, which then diffuse in the tumor microenvironment. The releaseof bioactive TNF has been shown to occur within 24-48 hours aftertargeting.

The preparation of cytokines in the form of liposomes can improve thebiological activity thereof. It has, in fact, been observed thatacylation of the TNF amino groups induces an increase in itshydrophobicity without loss of biological activity in vitro.Furthermore, it has been reported that TNF bound to lipids hasunaffected cytotoxicity in vitro, immunomodulating effects and reducedtoxicity in vivo.

Encapsulation of alpha v beta 3 L-TNF in liposomes could be another wayto improve, in qualitative terms, its biological profile. Thefeasibility of this approach was suggested by the observation thatacylation of some amino groups of TNF leads to an increase of itshydrophobicity without loss of biological activity in vitro. Thisfinding has been exploited to easily integrate TNF into lipid vesicles.Such lipid-bound TNF has been reported to possess unchanged in vitrocytotoxicity on tumour cells and immunomodulatory effects, while havingless toxic effects in vivo (48, (49).

Derivatisation of alphav beta3L-TNF with polyethylene glycol(pegylation) could be considered a preferred choice for prolonging itshalf life.

In many instances, the measured half-life of TNF in vivo, may be moreapparent than real. Thus, it was observed that this parameter is highlydependent on the administered dose and a disproportionate prolongationof half-life was observed at increasing doses of TNF (50). Oneexplanation for this phenomenon is that, at low doses, TNF isefficiently bound by soluble, circulating TNFR (51). Such soluble TNFRincrease rapidly in the serum of patients systemically treated with TNF(52) and arise by proteolytic cleavage from surface-bound receptors. TNFbound to circulating TNFR may escape detection in most assays commonlyused for the measurement of TNF levels. Above a threshold level at whichall soluble TNFR, both basal as well as TNF-induced, become saturated,measurements start to detect unbound, circulating TNF therebyreflecting, more accurately, the effective in vivo half-life of TNF.

It is clear that pegylation of TNF is not expected to obviate thisscavenging effect of TNFR and, thus, any approach aimed at prolongingthe half-life of TNF and, more generally, at reducing the doses of TNFto be administered, must deal with the fact that, in order to be active,TNF levels in vivo have to exceed the binding capacity of soluble,circulating TNFR. However one possibility to cope with this problem isto mutagenize CD13L-TNF to reduce its ability to interact with naturalTNF receptors, thus enablig higher doses to be administered.

Combined Approach

One of the earliest approaches that has been pursued to achieve a morefavourable therapeutic index for systemically administered TNF has beento combine TNF with other agents. The hope was to end up withtherapeutic protocols allowing to administer lower doses of TNF which,while preserving anti-tumour activity, had less systemic toxic effects.This rationale was highly speculative because it was not possible toexclude that such protocols would have ended up with a synergisticeffect also as regards toxicity and, therefore, with a therapeutic indexidentical to that observed with TNF alone. In fact, in all instances inwhich such combination therapy protocols have been studied in humans, itis the latter situation that has proven to be true.

One of these approaches that has been studied most intensively, is thecombined use of TNF and IFN-γ (36, 37), particularly because of thesynergism of action on endothelial cells of these cytokines. The secondapproach is the combination with chemotherapy.

Protocols combining TNF and some of the compounds described to synergisewith TNF have been studied in some experimental tumour models.Unfortunately, this treatment was accompanied by increased systemictoxicity.

Targeted delivery of TNF to tumor vessels is an approach that has beenrecently pursued to increase the therapeutic index of TNF. WO01/61017describes a TNF derivative with improved therapeutic index prepared bycoupling TNF with a ligand of aminopeptidase N (CD13), a membranepreotease expressed in tumor vessels. This cytokine interacts in a verycomplex manner with CD13 and TNF-receptors to selectively activate atlow doses tumor endothelial cells. Given the synergistic effect of TNFand IFN-γ on endothelial cells it would be advisable to targetendothelial cells with both cytokines conjugated to CD 13 ligands.However, one might expect that these modified cytokines compete for thesame receptor (CD13) on endothelial cells leading to loss of targetingand activity. WO01/61017 teaches how to prepare conjugates of thiscytokine with CD13 ligands, e.g. NGR-TNF and NGR-IFN-γ. Experimentscarried out in our laboratory based on administration of TNF and IFN-γboth conjugated to a CD13 ligand (CNGRC) showed that indeed when thesemodified cytokines are injected in animal models their therapeuticactivity is lower than when given alone, presumably because they competefor the same targeting receptor.

We have now found that these cytokines can be targeted to vesselswithout cross-interference in binding by, for example, targeting TNF toa tumor vascular receptor different to CD13 and IFN-γ to CD13 (e.g. bycoupling it to CNGRC peptide) or vice versa.

In this preferred embodiment of the invention, TNF is coupled to ligandsof alpha v beta 3, such as peptides containing the CRGDC motif Thus inone preferred embodiment of the present invention there is provided thecombined use of avb3L-IFN-γ derivative with CD13 ligand-TNF. In anotherpreferred embodiment there is provided the combined use of avb3L-TNFderivative with CD13 ligand-IFN-γ.

Polynucleotides

Polynucleotides for use in the invention comprise nucleic acid sequencesencoding the polypeptide conjugate of the invention. It will beunderstood by a skilled person that numerous different polynucleotidescan encode the same polypeptide as a result of the degeneracy of thegenetic code. In addition, it is to be understood that skilled personsmay, using routine techniques, make nucleotide substitutions that do notaffect the polypeptide sequence encoded by the polynucleotides of theinvention to reflect the codon usage of any particular host organism inwhich the polypeptides of the invention are to be expressed.

Polynucleotides of the invention may comprise DNA or RNA. They may besingle-stranded or double-stranded. They may also be polynucleotideswhich include within them synthetic or modified nucleotides. A number ofdifferent types of modification to oligonucleotides are known in theart. These include methylphosphonate and phosphorothioate backbones,addition of acridine or polylysine chains at the 3′ and/or 5′ ends ofthe molecule. For the purposes of the present invention, it is to beunderstood that the polynucleotides described herein may be modified byany method available in the art. Such modifications may be carried outin order to enhance the in vivo activity or life span of polynucleotidesof the invention.

Nucleotide Vectors

Polynucleotides of the invention can be incorporated into a recombinantreplicable vector. The vector may be used to replicate the nucleic acidin a compatible host cell. Thus in a further embodiment, the inventionprovides a method of making polynucleotides of the invention byintroducing a polynucleotide of the invention into a replicable vector,introducing the vector into a compatible host cell, and growing the hostcell under conditions which bring about replication of the vector. Thevector may be recovered from the host cell. Suitable host cells includebacteria such as E. coli, yeast, mammalian cell lines and othereukaryotic cell lines, for example insect Sf9 cells.

Preferably, a polynucleotide of the invention in a vector is operablylinked to a control sequence that is capable of providing for theexpression of the coding sequence by the host cell, i.e. the vector isan expression vector. The term “operably linked” means that thecomponents described are in a relationship permitting them to functionin their intended manner. A regulatory sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under condition compatible with the controlsequences.

The control sequences may be modified, for example by the addition offurther transcriptional regulatory elements to make the level oftranscription directed by the control sequences more responsive totranscriptional modulators.

Vectors of the invention may be transformed or transfected into asuitable host cell as described below to provide for expression of aprotein of the invention. This process may comprise culturing a hostcell transformed with an expression vector as described above underconditions to provide for expression by the vector of a coding sequenceencoding the protein, and optionally recovering the expressed protein.

The vectors may be for example, plasmid or virus vectors provided withan origin of replication, optionally a promoter for the expression ofthe said polynucleotide and optionally a regulator of the promoter. Thevectors may contain one or more selectable marker genes, for example anampicillin resistance gene in the case of a bacterial plasmid or aneomycin resistance gene for a mammalian vector. Vectors may be used,for example, to transfect or transform a host cell.

Control sequences operably linked to sequences encoding the protein ofthe invention include promoters/enhancers and other expressionregulation signals. These control sequences may be selected to becompatible with the host cell for which the expression vector isdesigned to be used in. The term “promoter” is well-known in the art andencompasses nucleic acid regions ranging in size and complexity fromminimal promoters to promoters including upstream elements andenhancers.

The promoter is typically selected from promoters which are functionalin mammalian cells, although prokaryotic promoters and promotersfunctional in other eukaryotic cells may be used. The promoter istypically derived from promoter sequences of viral or eukaryotic genes.For example, it may be a promoter derived from the genome of a cell inwhich expression is to occur. With respect to eukaryotic promoters, theymay be promoters that function in a ubiquitous manner (such as promotersof a-actin, b-actin, tubulin) or, alternatively, a tissue-specificmanner (such as promoters of the genes for pyruvate kinase).Tissue-specific promoters specific for certain cells may also be used.They may also be promoters that respond to specific stimuli, for examplepromoters that bind steroid hormone receptors. Viral promoters may alsobe used, for example the Moloney murine leukaemia virus long terminalrepeat (MMLV LTR) promoter, the rous sarcoma virus (RSV) LTR promoter orthe human cytomegalovirus (CMV) IE promoter.

It may also be advantageous for the promoters to be inducible so thatthe levels of expression of the heterologous gene can be regulatedduring the life-time of the cell. Inducible means that the levels ofexpression obtained using the promoter can be regulated.

In addition, any of these promoters may be modified by the addition offurther regulatory sequences, for example enhancer sequences. Chimericpromoters may also be used comprising sequence elements from two or moredifferent promoters described above.

Host Cells

Vectors and polynucleotides of the invention may be introduced into hostcells for the purpose of replicating the vectors/polynucleotides and/orexpressing the proteins of the invention encoded by the polynucleotidesof the invention. Although the proteins of the invention may be producedusing prokaryotic cells as host cells, it is preferred to use eukaryoticcells, for example yeast, insect or mammalian cells, in particularmammalian cells.

Vectors/polynucleotides of the invention may introduced into suitablehost cells using a variety of techniques known in the art, such astransfection, transformation and electroporation. Wherevectors/polynucleotides of the invention are to be administered toanimals, several techniques are known in the art, for example infectionwith recombinant viral vectors such as retroviruses, herpes simplexviruses and adenoviruses, direct injection of nucleic acids andbiolistic transformation.

Protein Expression and Purification

Host cells comprising polynucleotides of the invention may be used toexpress proteins of the invention. Host cells may be cultured undersuitable conditions which allow expression of the proteins of theinvention. Expression of the proteins of the invention may beconstitutive such that they are continually produced, or inducible,requiring a stimulus to initiate expression. In the case of inducibleexpression, protein production can be initiated when required by, forexample, addition of an inducer substance to the culture medium, forexample dexamethasone or IPTG.

Proteins of the invention can be extracted from host cells by a varietyof techniques known in the art, including enzymatic, chemical and/orosmotic lysis and physical disruption.

Administration

Proteins of the invention may preferably be combined with variouscomponents to produce compositions of the invention. Preferably thecompositions are combined with a pharmaceutically acceptable carrier,diluent or excipient to produce a pharmaceutical composition (which maybe for human or animal use). Suitable carriers and diluents includeisotonic saline solutions, for example phosphate-buffered saline.Details of excipients may be found in The Handbook of PharmaceuticalExcipients, 2nd Edn, Eds Wade & Weller, American PharmaceuticalAssociation. The composition of the invention may be administered bydirect injection. The composition may be formulated for parenteral,intramuscular, intravenous, subcutaneous, intraocular, oral ortransdermal administration.

The conjugate may typically be administered in a doasge of about 1 to 10mg.

The composition may be formulated such that administration daily, weeklyor monthly will provide the desired daily dosage. It will be appreciatedthat the composition may be conveniently formulated for administratedless frequently, such as every 2, 4, 6, 8, 10 or 12 hours.

Polynucleotides/vectors encoding polypeptide components may beadministered directly as a naked nucleic acid construct, preferablyfurther comprising flanking sequences homologous to the host cellgenome.

Uptake of naked nucleic acid constructs by mammalian cells is enhancedby several known transfection techniques for example those including theuse of transfection agents. Example of these agents include cationicagents (for example calcium phosphate and DEAE-dextran) and lipofectants(for example lipofectam™ and transfectam™). Typically, nucleic acidconstructs are mixed with the transfection agent to produce acomposition.

Preferably the polynucleotide or vector of the invention is combinedwith a pharmaceutically acceptable carrier or diluent to produce apharmaceutical composition. Suitable carriers and diluents includeisotonic saline solutions, for example phosphate-buffered saline. Thecomposition may be formulated for parenteral, intramuscular,intravenous, subcutaneous, intraocular or transdermal administration.

The routes of administration and dosage regimens described are intendedonly as a guide since a skilled practitioner will be able to determinereadily the optimum route of administration and dosage regimens for anyparticular patient and condition.

Viral Vectors

In a preferred embodiment the conjugate is administered using a viralvector, more preferably a retroviral vector.

Retroviruses

The retroviral vector for use the present invention may be derived fromor may be derivable from any suitable retrovirus. A large number ofdifferent retroviruses have been identified. Examples include: murineleukemia virus (MLV), human immunodeficiency virus (HIV), simianimmunodeficiency virus, human T-cell leukemia virus (HTLV), equineinfectious anaemia virus (EIAV), mouse mammary tumour virus (MMTV), Roussarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murineleukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV),Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus(A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avianerythroblastosis virus (AEV). A detailed list of retroviruses may befound in Coffin et al., 1997, “retroviruses”, Cold Spring HarbourLaboratory Press Eds: J M Coffin, S M Hughes, H E Varmus pp 758-763.

Details on the genomic structure of some retroviruses may be found inthe art. By way of example, details on HIV and Mo-MLV may be found fromthe NCBI Genbank (Genome Accession Nos. AF033819 and AF033811,respectively).

Retroviruses may be broadly divided into two categories: namely,“simple” and “complex”. Retroviruses may even be further divided intoseven groups. Five of these groups represent retroviruses with oncogenicpotential. The remaining two groups are the lentiviruses and thespumaviruses. A review of these retroviruses is presented in Coffin etal., 1997 (ibid).

The lentivirus group can be split even further into “primate” and“on-primate”. Examples of primate lentiviruses include humanimmunodeficiency virus (HIV), the causative agent of humanauto-immunodeficiency syndrome (AIDS), and simian immunodeficiency virus(SIV). The non-primate lentiviral group includes the prototype “slowvirus” visna/maedi virus (VMV), as well as the related caprinearthritis-encephalitis virus (CAEV), equine infectious anaemia virus(EIAV) and the more recently described feline immunodeficiency virus(FIV) and bovine immunodeficiency virus (BIV).

This invention also relates to the use of vectors for the delivery of aconjugate in the form of a nucleotide sequence to a haematopoietic stemcell (HSC).

Gene transfer involves the delivery to target cells, such as HSCs, of anexpression cassette made up of one or more nucleotide sequences and thesequences controlling their expression. This can be carried out ex vivoin a procedure in which the cassette is transferred to cells in thelaboratory and the modified cells are then administered to a recipient.Alternatively, gene transfer can be carried out in vivo in a procedurein which the expression cassette is transferred directly to cells withinan individual. In both strategies, the transfer process is usually aidedby a vector that helps deliver the cassette to the appropriateintracellular site.

Bone marrow has been the traditional source of HSCs for transduction,more recent studies have suggested that peripheral blood stem cells orcord blood cells may be equally good or better target cells (Cassel etal 1993 Exp Hematol 21: 585-591; Bregni et al 1992 Blood 80: 1418-1422;Lu et al 1993 J Exp Med 178: 2089-2096).

Further Anticancer Agents

The conjugate of the present invention may be used in combination withone or more other active agents, such as one or more cytotoxic drugs.Thus, in one aspect of the present invention the method furthercomprises administering another active pharmaceutical ingredient, suchas a cytotoxic drug, either in combined dosage form with the conjugateor in a separate dosage form. Such separate cytotoxic drug dosage formmay include solid oral, oral solution, syrup, elixir, injectable,transdermal, transmucosal, or other dosage form. The conjugate and theother active pharmaceutical ingredient can be combined in one dosageform or supplied in separate dosage forms that are usable together orsequentially.

Examples of cytotoxic drugs which may be used in the present inventioninclude: the alkylating drugs, such as cyclophosphamide, ifospfamide,chlorambucil, melphalan, busulfan, lomustine, carmustine, chlormethhine(mustine), estramustine, treosulfan, thiotepa, mitobronitol; cytotoxicantibiotics, such as doxorubicin, epirubicin, aclarubicin, idarubicin,daunorubicin, mitoxantrone (mitozantrone), bleomycin, dactinomycin andmitomycin; antimetabolites, such as methotrexate, capecitabine,cytarabine, fludarabine, cladribine, gemcitabine, fluorouracil,raltitrexed, mercaptopurine, tegafur and tioguanine; vinca alkaloids,such as vinblastine, vincristine, vindesine and vinorelbine, andetoposide; other neoplastic drugs, such as amsacrine, altretamine,crisantaspase, dacarbazine and temozolomide, hydroxycarbamide(hydroxyurea), pentostatin, platinum compounds including: carboplatin,cisplatin and oxaliplatin, porfimer sodium, procarbazine, razoxane,taxanes including: docetaxel and paclitaxel, topoisomerase I inhibitorsincluding: irinotecan and topotecan, trastuzumab, and tretinoin.

In a preferred embodiment the further cytotoxic drug is doxorubicin ormelphalan.

The conjugate of the present invention can also be used to use thepermeability of tumor cells and vessels to compounds for diagnosticpurposes. For instance, the conjugate can be used to increase the tumoruptake of radiolabelled antibodies or hormones (tumor-imaging compounds)in radioimmunoscintigraphy or radiotherapy of tumors.

FIGURES AND EXAMPLES

The present invention will further be described by reference to thefollowing non-limiting Examples and Figure in which:

FIG. 1 illustrates the characterization of the therapeutic and toxicactivity of TNF and RGD-TNF in combination with NGR-IFN in the T/SAmouse mammary adenocarcinoma model. In more detail it shows that theantitumor activity of RGD-mTNF in combination with NGR-mIFN-γ isstronger than that of mTNF administered in combination with NGR-mIFN-γor that of NGR-mIFN-γ alone. These results indicate that targeteddelivery of TNF and IFN-γ to different receptors on the tumorvasculature can produce synergistic effects.

EXAMPLES Example I

Preparation of TNF and RGD-TNF.

Murine recombinant TNF and ACDCRGDCFCG-TNF (RGD-TNF) were produced bycytoplasmic cDNA expression in E. coli. The cDNA coding for murineMet-TNF₁₋₁₅₆ (66) was prepared by reverse transcriptase-polymerase chainreaction (RT-PCR) on mRNA isolated from lipopolysaccharide-stimulatedmurine RAW-264.7 monocyte-macrophage cells, using5′-CTGGATCCTCACAGAGCAATGACTCCAAAG-3′ and5′-TGCCTCACATATGCTCAGATCATCTTCTC-3′, as 3′ and 5′ primers.

The amplified fragment was digested with Nde I and Bam HI (New EnglandBiolabs, Beverley, Mass.) and cloned in pET-I lb (Novagen, Madison,Wis.), previously digested with the same enzymes (pTNF).

The cDNA coding for ACDCRGDCFCG-TNF₁₋₁₅₆ was amplified by PCR on pTNF,using 5′-TGCAGATCATATGGCTTGCGACTGCCGTGGTGACTGCTTCTGCGGTCTCAGAT CATCTTCTC3′ as 5′ primer, and the above 3′ primer.

The amplified fragment was digested and cloned in pET-11b as describedabove and used to transform BL21(DE3) E. coli cells (Novagen). Theexpression of TNF and RGD-TNF was induced withisopropyl-□-D-tiogalactoside, according to the pET11b manufacturer'sinstruction. Soluble TNF and RGD-TNF were recovered from two-litercultures by bacterial sonication in 2 mM etilendiaminetetracetic acid,20 mM Tris-HCl, pH 8.0, followed by centrifugation (15000×g, 20 min, 4°C.). Both extracts were mixed with ammonium sulfate (25% of saturation),left for 1 h at 4° C., and further centrifuged, as above. The ammoniumsulfate in the supernatants was then brought to 65% of saturation, leftat 4° C. for 24 h and further centrifuged. Each pellet was dissolved in200 ml of 1 M ammonium sulfate, 50 mM Tris-HCl, pH 8.0, and purified byhydrophobic interaction chromatography on Phenyl-Sepharose 6 Fast Flow(Pharmacia-Upjohn) (gradient elution, buffer A: 50 mM sodium phosphate,pH 8.0, containing 1 M ammonium sulfate; buffer B: 20% glycerol, 5%methanol, 50 mM sodium phosphate, pH 8.0). Fractions containing TNFimmunoreactive material (by western blotting) were pooled, dialyzedagainst 2 mM etilendiaminetetracetic acid, 20 mM Tris-HCl, pH 8.0 andfurther purified by ion exchange chromatography on DEAE-Sepharose FastFlow (Pharmacia-Upjohn) (gradient elution, buffer A: 20 mM Tris-HCl, pH8.0; buffer B: 1 M sodium chloride, 20 mM Tris-HCl, pH 8.0). Fractionscontaining TNF-immunoreactivity were pooled and purified by gelfiltration chromatography on Sephacryl-S-300 HR (Pharmacia-Upjohn),pre-equilibrated and eluted with 150 mM sodium chloride, 50 mM sodiumphosphate buffer, pH 7.3 (PBS). Fractions corresponding to 40000-50000Mr products were pooled, aliquoted and stored frozen at −20° C. Allsolutions employed in the chromatographic steps were prepared withsterile and endotoxin-free water (Salf, Bergamo, Italy).

The molecular weight of purified TNF and RGD-TNF was measured byelectrospray mass spectrometry, as described (65). The protein contentwas measured using a commercial protein assay kit (Pierce, Rockford,Ill.).

Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) andwestern blot analysis were carried out using 12.5 or 15% polyacrylamidegels, by standard procedures.

Non reducing SDS-PAGE of TNF showed a single band of 17-18 kDa, asexpected for monomeric TNF. At variance, non reducing SDS-PAGE andwestern blot analysis of RGD-TNF showed different immunoreactive formsof 18, 36 and 50 kDa, likely corresponding to monomers, dimers andtrimers. Under reducing conditions most of the 50 and 36 kDa bands wereconverted into the 18 kDa form, pointing to the presence of RGD-TNFmolecules with interchain disulfide bridges. The 18 kDa band accountedto about ½ of the total material. These electrophoretic patterns suggestthat RGD-TNF was a mixture of trimers made up by three monomericsubunits with correct intra-chain disulfides (10-20%) and the remainingpart mostly by trimers with one or more interchain disulfides.

The molecular mass of TNF and RGD-TNF monomers were 17386.1±2.0 Da and18392.8 Da, respectively, by electrospray mass spectrometry. Thesevalues correspond very well to the mass expected for Met-TNF₁₋₁₅₆(17386.7 Da) and for ACDCRGDCFCG-TNF₁₋₁₅₆ (18392.9 Da).

Example II

In Vitro Cytotoxic Activity of TNF and RGD-TNF.

The bioactivity of TNF and RGD-TNF was estimated by standard cytolyticassay based on L-M mouse fibroblasts (ATCC CCL1.2) as described (67).The cytolytic activity of TNF and NGR-TNF on RMA-T cells was tested inthe presence of 30 ng/ml actinomycin D (68). Each sample was analyzed induplicate, at three different dilutions. The results are expressed asmean±SD of two-three independent assays.

The in vitro cytotoxic activity of TNF and RGD-TNF was (1.2±0.14)×10⁸units/mg and (1.7±1)×10⁸ units/mg, respectively, by standard cytolyticassay with L-M cells. These results indicate that the ACDCRGDCFCGmoieties in the RGD-TNF molecule does not prevent folding,oligomerizazion and binding to TNF receptors.

In a previous study we showed that RMA-T cells can be killed by TNF inthe presence of 30 ng/ml actinomycin D, whereas in the absence oftranscription inhibitors these cells are resistant to TNF, even afterseveral days of incubation (68). The in vitro cytotoxic activity ofRGD-TNF on RMA-T cells in the presence of actinomycin D was(1.6+1.3)×10⁸ units/mg, as measured using TNF ((1.2±0.14)×10⁸ units/mg)as a standard.

Example III

Characterization of the Therapeutic and Toxic Activity of TNF andRGD-TNF.

The Rauscher virus-induced RMA lymphoma of C57BL/6 origin (69) weremaintained in vitro in RPMI 1640, 5% fetal bovine serum (FBS), 100 U/mlpenicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin B, 2 mMglutamine and 50 μM 2-mercaptoethanol. RMA-T was derived from the RMAcell line by transfection with a construct encoding the Thy 1.1 alleleand cultured as described Moro, 1997 #28].

T/SA mouse mammary adenocarcinoma cells were cultured as described ( ).

In vivo studies on animal models were approved by the Ethical Committeeof the San Raffaele H Scientific Institute and performed according tothe prescribed guidelines. C57BL/6 (Charles River Laboratories, Calco,Italy) (16-18 g) were challenged with 5×10⁴ RMA-T or TSA living cells,respectively, s.c. in the left flank. Ten-twelve days after tumorimplantation, mice were treated, i.p., with 250 μl TNF or RGD-TNFsolutions, diluted with endotoxin-free 0.9% sodium chloride. Preliminaryexperiments showed that the anti-tumor activity was not changed by theaddition of human serum albumin to TNF and RGD-TNF solutions, as acarrier. Each experiment was carried out with 5 mice per group. Thetumor growth was monitored daily by measuring the tumor size withcalipers. The tumor area was estimated by calculating r₁×r₂ π, whereastumor volume was estimated by calculating r₁×r₂×r₃×4/3 π, where r₁ andr₂ are the longitudinal and lateral radii, and r₃ is the thickness oftumors protruding from the surface of normal skin. Animals were killedbefore the tumor reached 1.0-1.3 cm diameter. Tumor sizes are shown asmean±SE (5-10 animals per group) and compared by t-test.

The anti-tumor activity and toxicity of RGD-TNF were compared to thoseof TNF using the RMA-T lymphoma and the T/SA models in C57BL6 mice.

Murine TNF administered to animals bearing established s.c. RMA-Ttumors, causes 24 h later a reduction in the tumor mass and haemorragicnecrosis in the central part of the tumor, followed by a significantgrowth delay for few days (71). A single treatment with TNF does notinduce complete regression of this tumor, even at doses close to theLD50, as living cells remaining around the necrotic area restart to growfew days after treatment. In a first set of experiments we investigatedthe effect of various doses (i.p.) of TNF or RGD-TNF on animal survival.To avoid excessive suffering, the animals were killed when the tumordiameter was greater than 1-1.3 cm. The lethality of TNF and RGD-TNF, 3days after treatment, was different (LD50, 6 μg and 12 μg, respectively)whereas their anti-tumor activity was markedly different (Table 1). Forinstance, 1 of μg of RGD-TNF delayed the tumor growth more efficientlythen 2 μg of TNF. Interestingly, some animals were cured with 16 μg ofRGD-TNF whereas no animals at all were cured with TNF. Cured animalsrejected further challenges with tumorigenic doses of either RMA-T orwild-type RMA cells, suggesting that a single treatment with RGD-TNF wasable to induce protective immunity.

Thus, the calculated efficacy/toxicity ratio of RGD-TNF under theseconditions is 4 times greater than that of TNF. Considering that theform with correct disulfide bridges in the RGD-TNF preparation is about10-20% one may calculate that the therapeutic index of RGD-TNF is 2040%higher than that of TNF.

Moreover, RGD-TNF can induce protective immune responses moreefficiently than TNF.

Since RMA-T cells do not express the alpha v integrin (by FACS with ananti-alpha v antibody) while endothelial cells can express this integrinthe results suggest that the mechanism of action is based on targetingcells other than tumor cells, e.g. endothelial cells. TABLE 1 Survival(%) of RMA-Thy 1.1 lymphoma bearing mice treated 12 days after tumorimplantation with TNF or RGD-TNF (i.v.) Survival (%)^(a) Day Day AnimalsDose Day Day Day Day 90 115 Day Treatment (n) (μg i.v.) 14 22 32 37 (2ndch.)^(b) (3rd ch.)^(b) 160 none 9 0 100 0 TNF 9 1 100 22 0 8 2 100 37 010 4 100 70 30 10 0 10 8 0 10 16 0 total 47 RGD-TNF 10 1 100 30 20 0 7 2100 85 15 0 10 4 100 50 10 10 0 10 8 90 90 30 10 0 10 16 30 30 20 20 2020 20 total 47^(a)The cumulative results of two independent experiments (5 animals pergroup of treatment) are shown. Animals with ascitic tumors were notincluded in the study.^(b)Surviving animals were re-challanged with 50.000 RMA-T at day 90followed by 50.000 RMA cells at day 115, respectively. At the same timefive normal animals were treated with the same cells to check thetumorigenicity of the injected dose. All control animals developed atumor within 10 days.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are apparent to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

REFERENCES

-   1. Corti A, et al. Biochemical Journal. 1992; 284: 905-10.-   2. Tartaglia L A, et al. Proceedings of the National Academy of    Sciences of the United States of America. 1991; 88: 9292-6.-   3. Espevik T, et al. Journal of Experimental Medicine. 1990; 171:    415-26.-   4. Loetscher H, et al. Journal of Biological Chemistry. 1993; 268:    26350-7.-   5. Van Ostade X, et al. European Journal of Biochemistry. 1994; 220:    771-779.-   6. Barbara J A, et al. EMBO Journal. 1994; 13: 843-50.-   7. Engelmann H, et al. J. Biol. Chem. 1990; 265: 14497.-   8. Bigda J, et al. Journal of Experimental Medicine. 1994; 180:    445-60.-   9. Tartaglia L A, et al. Journal of Biological Chemistry. 1993; 268:    18542-8.-   10. Vandenabeele P, et al. Journal of Experimental Medicine. 1992;    176: 1015-24.-   11. Naume B, et al. Journal of Immunology. 1991; 146: 3045-8.-   12. Grell M, et al. Cell. 1995; 83: 793-802.-   13. Carswell E A, et al. Proc. Natl. Acad. Sci. USA. 1975; 72:    3666-70.-   14. Helson L, et al. Nature. 1975; 258: 731-732.-   15. Tracey K J and Cerami A. Annual Review of Cell Biology. 1993; 9:    31743.-   16. Elliott M J, et al. International Journal of Immunopharmacology.    1995; 17: 141-5.-   17. Palladino M A, Jr., et al. Journal of Immunology. 1987; 138:    4023-32.-   18. Clauss M, et al. Journal of Biological Chemistry. 1990; 265:    7078-83.-   19. Nawroth P P and Stem D M. Journal of Experimental Medicine.    1986; 163: 740-5.-   20. Clauss M, et al. Journal of Experimental Medicine. 1990; 172:    1535-45.-   21. McIntosh J K, et al. Cancer Research. 1990; 50: 2463-9.-   22. Meulders Q, et al. Kidney International. 1992; 42: 327-34.-   23. van de Wiel P A, et al. Immunopharmacology. 1992; 23: 49-56.-   24. Nawroth P, et al. Journal of Experimental Medicine. 1988; 168:    637-47.-   25. Stryhn Hansen A, et al. European Journal of Immunology. 1993;    23: 2358-64.-   26. Taylor A. FASEB Journal. 1993; 7: 290-8.-   27. Shipp M A and Look A T. Blood. 1993; 82: 1052-70.-   28. Fraker D L, Alexander H R and Pass H I: Biologic therapy with    TNF: systemic administration and isolation-perfusion. in Biologic    therapy of cancer: principles and practice. V. De Vita, S. Hellman    and S. Rosenberg, ed. J.B. Lippincott Company:Philadelphia.    1995.329-345.-   29. Fiers W: Biologic therapy with TNF: preclinical studies. in    Biologic therapy of cancer: principles and practice. V. De Vita, S.    Hellman and S. Rosenberg, ed. J.B. Lippincott Company:Philadelphia.    1995.295-327.-   30. Sidhu R S and Bollon A P. Pharmacological Therapy. 1993; 57:    79-128.-   31. Hieber U and Heim M E. Oncology. 1994; 51: 142-53.-   32. Lienard D, et al. World Journal of Surgery. 1992; 16: 234-40.-   33. Hill S, et al. British Journal of Surgery. 1993; 80: 995-7.-   34. Eggermont A M, et al. Annals of Surgery. 1996; 224: 756-65.-   35. Schraffordt Koops H, et al. Radiotherapy and Oncology. 1998; 48:    1-4.-   36. Williamson B D, et al. Proceedings of the National Academy of    Sciences of the United States of America. 1983; 80: 5397-401.-   37. Fransen L, et al. European Journal of Cancer & Clinical    Oncology. 1986; 22: 419-26.-   38. Ruff M R and Gifford G E: Tumor Necrosis Factor. in    Lymphokines. E. Pick, ed. Academic Press:New York. 1981.235-272.-   39. Beyaert R, et al. Cancer Research. 1993; 53: 2623-30.-   40. Beyaert R, et al. Proceedings of the National Academy of    Sciences of the United States of America. 1989; 86: 9494-8.-   41. Balkwill F R, et al. Cancer Research. 1986; 46: 3990-3.-   42. Schiller J H, et al. Cancer. 1992; 69: 562-71.-   43. Jones A L, et al. Cancer Chemotherapy & Pharmacology. 1992; 30:    73-6.-   44. Brouckaert P, et al. Lymphokine & Cytokine Research. 1992; 11:    193-6.-   45. Van Ostade X, et al. Nature. 1993; 361: 266-9.-   46. Van Zee K J, et al. Journal of Experimental Medicine. 1994; 179:    1185-91.-   47. Bartholeyns J, et al. Infection & Immunity. 1987; 55: 2230-3.-   48. Debs R J, et al. Journal of Immunology. 1989; 143: 1192-7.-   49. Debs R J, et al. Cancer Research. 1990; 50: 375-80.-   50. Kimura K, et al. Cancer Chemotherapy & Pharmacology. 1987; 20:    223-9.-   51. Aderka D, et al. Cancer Research. 1991; 51: 5602-7.-   52. Lantz M, et al. Cytokine. 1990; 2: 402-6.-   53. Hoogenboom H R, et al. Molecular Immunology. 1991; 28: 1027-37.-   54. Yang J, et al. Human Antibodies & Hybridomas. 1995; 6: 129-36.-   55. Yang J, et al. Molecular Immunology. 1995; 32: 873-81.-   56. Pasqualini R, et al. Nature Biotechnology. 1997; 15: 542-6.-   57. Koivunen E, et al. Nature Biotechnology. 1999; 17: 768-774.-   58. Brekken R A, et al. Cancer Research. 1998; 58: 1952-1959.-   59. Goodwin D A. Journal of Nuclear Medicine. 1995; 36: 876-9.-   60. Paganelli G, et al. Cancer Research. 1991; 51: 5960-6.-   61. Modorati G, et al. British Journal of Ophtalmology. 1994; 78:    19-23.-   62. Colombo P, et al. Journal of Endocrinological Investigation.    1993; 16: 841-3.-   63. Paganelli G, Magnani P, Siccardi A and Fazio F: Clinical    application of the avidin-biotin system for tumor targeting. in    Cancer therapy with radiolabeled antibodies. D. Goldenberg, ed. CRC    Press:Boca Raton. 1995.239-253.-   64. Robert B, et al. Cancer Research. 1996; 56: 4758-4765.-   65. Corti A, et al. Cancer Research. 1998; 58: 3866-3872.-   66. Pennica D, et al. Proceedings of the National Academy of    Sciences of the United States of America. 1985; 82: 6060-4.-   67. Corti A, et al. Journal of Immunological Methods. 1994; 177:    191-198.-   68. Moro M, et al. Cancer Research. 1997; 57: 1922-8.-   69. Ljunggren H G and Karre K. Journal of Experimental Medicine.    1985; 162: 1745-59.-   70. Celik C, et al. Cancer Research. 1983; 43: 3507-10.-   71. Gasparri A, et al. Cancer Research. 1999; 59: 2917-23.-   72. Arap W, et al. Science. 1998; 279: 377-80.-   73. Talmadge J E, et al. Cancer Research. 1987; 47: 2563-70.-   74. Pfizemaier K, et al. Journal of Immunology. 1987; 138: 975-80.-   75. Asher A L, et al. Journal of Immunology. 1991; 146: 3227-34.-   76. Mizuguchi H, et al. Cancer Research. 1998; 58: 5725-30.-   77. Gasparri A, et al. Journal of Biological Chemistry. 1997; 272:    20835-43.

1. A conjugate of a cytokine and a tumor targeting moiety (TTM) with theprovisos that when the cytokine is TNF-α, TNF-β or IFN-γ, the TTM isother than a CD13 ligand; when the cytokine is IL-2 or IL-12, the TTM isother than an antibody to fibronectin; when the cytokine is TNF, the TTMis other than an antibody to the transferrin receptor; when the cytokineis TNF, IFN-γ or IL-2 the TTM is other than an antibody to the TAG72antigen; when the cytokine is IFN, the TTM is other than αvβ3 integrinligand and when the cytokine is TNF, the TTM is other than fibronectin.2. A conjugate according to claim 1 with the further proviso that whenthe cytokine is TNF-α or TNF-β, the TTM is other than a tumor specificantibody.
 3. A conjugate according to claim 1 with the further provisothat the conjugate is not biotinylated TNF.
 4. A conjugate according toclaim 1 wherein the cytokine is an inflammatory cytokine.
 5. A conjugateaccording to claim 1 wherein the cytokine is a chemotherapeuticcytokine.
 6. A conjugate according to any preceding claim 1 wherein thecytokine is TNFα, TNFβ, IFNα, IFNβ, IFNγ, IL-1, 2, 4, 6, 12, 15, EMAPII, vascular endothelial growth factor (VEGF), PDGF, PD-ECGF or achemokine.
 7. A conjugate according to claim 1 wherein the cytokine isTNF-α, TNF-β or IFN-γ.
 8. A conjugate according to claim 1 wherein theTTM is a tumor vasculature targeting moiety (TVTM).
 9. A conjugateaccording to claim 8 wherein the TVTM is a binding partner of a tumorvasculature receptor, marker or other extracellular component, such as apeptide which targets the tumor vasculature.
 10. A conjugate accordingto of claim 1 wherein the TTM is a binding partner of a tumor receptor,marker or other extracellular component.
 11. A conjugate according toclaim 1 wherein the TTM is an antibody or ligand, or a fragment thereof.12. A conjugate according to claim 1 wherein the TTM is contains the NGRor RGD motif, or is HIV-tat, Annexin V, Osteopontin, Fibronectin,Collagen Type I or IV, Hyaluronate, Ephrin, or is a binding partner tooncofetal fibronectin; or a fragment thereof.
 13. A conjugate accordingto claim 1 wherein the TTM contains the NGR motif.
 14. A conjugateaccording to claim 13 wherein the TTM is CNGRCVSGCAGRC, NGRAHA, GNGRG,cycloCVLNGRMEC, linear or cyclic CNGRC.
 15. A conjugate according toclaim 1 wherein the TTM contains the RGD motif.
 16. A conjugateaccording to claims 1 wherein the TTM is targeted to VEGFR, ICAM 1, 2 or3, PECAM-1, CD31, CD13, VCAM-1, Selectin, Act R11, ActRIIB, ActRI,ActRIB, CD44, aminopeptidase A, aminopeptidase N (CD13), αvβ3 integrin,αvβ5 integrin, FGF-1, 2, 3, or 4, IL-1R, EPHR, MMP, NG2, tenascin,oncofetal fibronectin, PD-ECGFR, TNFR, PDGFR or PSMA.
 17. A conjugateaccording to claim 1 as listed in Table A.
 18. A conjugate according toclaim 1 wherein the conjugate is in the form of a fusion protein.
 19. Aconjugate according to claim 1 wherein the conjugate is in the form ofnucleic acid.
 20. An expression vector comprising the nucleic acid ofclaim
 19. 21. A host cell transformed with the expression vector ofclaim
 20. 22. A method for preparing a conjugate comprising culturingthe host cell of claim 21 under conditions which provide for theexpression of the conjugate.
 23. A pharmaceutical composition comprisingthe conjugate of claim 1, together with a pharmaceutically acceptablecarrier, diluent or excipient.
 24. A pharmaceutical compositionaccording to claim 23 wherein the composition further comprises anotherantitumor agent or diagnostic tumor-imaging compound.
 25. Apharmaceutical composition according to claim 24 wherein the furtherantitumor agent is doxorubicin or melphalan.
 26. [canceled]
 27. A methodof treating or diagnosing cancer comprising administering to a patientin need of the same an effective amount of a conjugate as defined inclaim
 1. 28. A pharmaceutical composition comprising an effective amountof a conjugation product of TNF and a first TTM or a polynucleotideencoding the same, and an effevctive amount of IFN-γ and a second TTM ora polynucleotide encoding the same, wherein said first TTM and saidsecoond TTM compete for different receptors.
 29. A composition accordingto claim 27 together with a pharmaeutically acceptable carrier, diluentor excipient.
 30. A compostion according to claim 27 wherein said firstor said second TTM is a ligand of the CD13 receptor.
 31. A compositionaccording to claim 27 wherein said first or said second TTM contains theNGR motif.
 32. A composition according to claim 27 wherein said first orsaid second TTM is CNGRCVSGCAGRC, NGRAHA, GNGRG, cycloCVLNGRMEC, linearor cyclic CNGRC.
 33. A composition according to claim 27 wherein saidfirst or said second TTM is a ligand of the αvβ3 receptor.
 34. Acomposition according to claim 27 wherein said first or said second TTMcontains the RGD motif.
 35. A composition according to claim 27 whereinsaid first TTM is a ligand of the CD13 receptor and said second TTM is aligand of the αvβ3 receptor.
 36. A composition according to claim 27wherein said first TTM is a ligand of the αvβ3 receptor and said secondTTM is a ligand of the CD13 receptor.
 37. A conjugate according to claim2 with the further proviso that the conjugate is not biotinylated TNF.